We have been investigating the molecular and cellular mechanisms underlying the hormonal regulation of ovarian follicle growth and differentiation. By expressing recombinant FSH, LH and hCG and producing their mutants, we have designed long-acting agonists as well as deglycosylated antagonists of gonadotropins. We have also cloned human LH and FSH receptors and the expression of these proteins allows analysis of gonadotropin bioactivity in vitro. The extracellular ligand-binding domain of these receptors have been generated and found to be functional antagonists. Clinical syndromes of gain-of-function mutations for the LH receptor have been found in patients with familial male precocious puberty whereas loss-of-function mutations have been found to be the basis of Leydig cell hypoplasia. We are using bioinformatic tools and DNA microarray to analyze polypeptide hormones andtheir receptors in terms of ligand-receptor matching and paracrine interactions.
To assess if infusion of human bone marrow-derived stem cells (BMDSCs) could promote follicle development in patients with impaired ovarian functions.Experimental design.University research laboratories.Immunodeficient NOD/SCID female mice.Human BMDSCs were injected into mice with chemotherapy-induced ovarian damage and into immunodeficient mice xenografted with human cortex from poor-responder patients (PRs).Follicle development, ovulation, and offspring. Apoptosis, proliferation, and vascularization were evaluated in mouse and human ovarian stroma.Fertility rescue and spontaneous pregnancies were achieved in mice ovaries mimicking PRs and ovarian insufficiency, induced by chemotherapy, after BMDSC infusion. Furthermore, BMDSC treatment resulted in production of higher numbers of preovulatory follicles, metaphase II oocytes, 2-cell embryos, and healthy pups. Stem cells promoted ovarian vascularization and cell proliferation, along with reduced apoptosis. In xenografted human ovarian tissues from PRs, infusion of BMDSCs and their CD133+ fraction led to their engraftment close to follicles, resulting in promotion of follicular growth, increases in E2 secretion, and enhanced local vascularization.Our results raised the possibility that promoting ovarian angiogenesis by BMDSC infusion could be an alternative approach to improve follicular development in women with impaired ovarian function.NCT02240342.
View details for PubMedID 29576341
During each reproductive cycle, the ovary exhibits tissue remodelling and cyclic vasculature changes associated with hormonally regulated folliculogenesis, follicle rupture, luteal formation and regression. However, the relationships among different types of follicles and corpora lutea are unclear, and the role of ovarian vasculature in folliculogenesis and luteal dynamics has not been extensively investigated. Understanding of ovarian physiology and pathophysiology relies upon elucidation of ovarian morphology and architecture. This paper summarizes the literature on traditional approaches to the imaging of ovarian structures and discusses recent advances in ovarian imaging. Traditional in-vivo ultrasound, together with histological and electron microscopic approaches provide detailed views of the ovary at organ, tissue and molecular levels. However, in-vivo imaging is limited to antral and larger follicles whereas histological imaging is mainly two-dimensional in nature. Also discussed are emerging approaches in the use of near-infrared fluorophores to image follicles in live animals to detect preantral follicles as well as visualizing ovarian structures using CLARITY in fixed whole ovaries to elucidate three-dimensional interrelationships among follicles, corpora lutea and ovarian vasculature. Advances in ovarian imaging techniques provide new understanding of ovarian physiology and allow for the development of better tools to diagnose ovarian pathophysiology.
View details for PubMedID 29602728
Greatly reduced scattering in the second near-infrared (NIR-II) region (1000-1700 nm) opens up many new exciting avenues of bioimaging research, yet NIR-II fluorescence imaging is mostly implemented by using nontargeted fluorophores or wide-field imaging setups, limiting the signal-to-background ratio and imaging penetration depth due to poor specific binding and out-of-focus signals. A newly developed high-performance NIR-II bioconjugate enables targeted imaging of a specific organ in the living body with high quality. Combined with a home-built NIR-II confocal set-up, the enhanced imaging technique allows 900 µm-deep 3D organ imaging without tissue clearing techniques. Bioconjugation of two hormones to nonoverlapping NIR-II fluorophores facilitates two-color imaging of different receptors, demonstrating unprecedented multicolor live molecular imaging across the NIR-II window. This deep tissue imaging of specific receptors in live animals allows development of noninvasive molecular imaging of multifarious models of normal and neoplastic organs in vivo, beyond the traditional visible to NIR-I range. The developed NIR-II fluorescence microscopy will become a powerful imaging technique for deep tissue imaging without any physical sectioning or clearing treatment of the tissue.
View details for PubMedID 29446156
View details for PubMedCentralID PMC5931222
View details for Web of Science ID 000429928200152
Gonadotropins belong to the family of dimeric glycoprotein hormones and regulate gonadal physiology mediated by G protein-coupled, seven-transmembrane receptors. These glycoprotein hormones are widely used in the clinic to promote ovarian follicle development and for treating some cases of male infertility. We traced the co-evolution of dimeric gonadotropin hormones and their receptors, together with thyrotropin and its receptor. We updated recent findings on human genetic variants of these genes and their association with dizygotic twining, polycystic ovarian syndrome, primary ovarian insufficiency, male-limited precocious puberty and infertility. In addition to the known physiological roles of gonadotropin-receptor signaling in gonadal tissues, we also discussed emerging understanding of extra-gonadal functions of gonadotropins in bones and adipose tissues, together with recent advances in in vivo imaging of gonadotropin receptors in live animals. Recent development of gonadotropin receptor agonists and antagonists were summarized with an emphasis on the development of functional antagonists for FSH receptors to alleviate osteoporosis and obesity associated with menopause.
View details for PubMedID 29462242
The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P <0.01) between the length of the follicular capillaries and volume of the follicular wall.
View details for PubMedID 29286393
The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.
View details for DOI 10.1038/nature22313
View details for PubMedID 28467820
The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.
View details for DOI 10.1038/nature22313
View details for Web of Science ID 000400963800037
In vivo imaging of hormone receptors provides the opportunity to visualize target tissues under hormonal control in live animals. Detecting longer-wavelength photons in the second near-infrared window (NIR-II, 1000-1700 nm) region affords reduced photon scattering in tissues accompanied by lower autofluorescence, leading to higher spatial resolution at up to centimeter tissue penetration depths. Here, we report the conjugation of a small molecular NIR-II fluorophore CH1055 to a follicle stimulating hormone (FSH-CH) for imaging ovaries and testes in live mice. After exposure to FSH-CH, specific NIR-II signals were found in cultured ovarian granulosa cells containing FSH receptors. Injection of FSH-CH allowed live imaging of ovarian follicles and testicular seminiferous tubules in female and male adult mice, respectively. Using prepubertal mice, NIR-II signals were detected in ovaries containing only preantral follicles. Resolving earlier controversies regarding the expression of FSH receptors in cultured osteoclasts, we detected for the first time specific FSH receptor signals in bones in vivo. The present imaging of FSH receptors in live animals using a ligand-conjugated NIR-II fluorophore with low cell toxicity and rapid clearance allows the development of non-invasive molecular imaging of diverse hormonal target cells in vivo.
View details for DOI 10.1039/c6sc04897h
View details for Web of Science ID 000400553000048
View details for PubMedCentralID PMC5465568
Epithelial ovarian carcinomas account for more than 90% of human ovarian cancers and have become the primary cause of death for gynecological malignancies. Unlimited cell proliferation and resistance to cell apoptosis contribute to the development of ovarian cancers. However, the underlying mechanisms involved in these processes in epithelial ovarian carcinomas are yet poorly understood. In the present study, we examined the Hippo signaling gene expression and investigated the effects of Sphingosine 1-phosphate (S1P) on cell proliferation and the underlying mechanisms in human ovarian cancer cell lines, OVCAR3 and SKOV3. Our results demonstrate that S1P disrupts Hippo signaling by reducing YAP phosphorylation and increasing the expression of CCN1 and CCN2 in both ovarian cancer cells. Furthermore, the increase in CCN1/CCN2 expression contributes to the S1P-induced increase in cancer cell proliferation.
View details for DOI 10.18632/oncotarget.15677
View details for PubMedID 28460443
Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 10(5)-fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.
View details for DOI 10.1038/srep44810
View details for PubMedID 28333125
Recently, two patients with primary ovarian insufficiency (POI) delivered healthy babies after in vitro activation (IVA) treatment followed by auto-transplantation of frozen-thawed ovarian tissues.This study sought to report the first case of live birth after IVA treatment following fresh ovarian tissue grafting in patients with POI, together with monitoring of follicle development and serum hormonal changes.This was a prospective observational cohort study.We performed IVA treatment in 14 patients with POI with mean age of 29 years, mean duration since last menses of 3.8 years, and average basal FSH level of 94.5 mIU/mL.Prior to IVA treatment, all patients received routine hormonal treatments with no follicle development. We removed one ovary from patients with POI and treated them with Akt stimulators. We improved upon early procedures by grafting back fresh tissues using a simplified protocol.In six of the 14 patients (43%), a total of 15 follicle development waves were detected, and four patients had successful oocyte retrieval to yield six oocytes. For two patients showing no spontaneous follicle growth, human menopausal gonadotropin treatment induced follicle growth at 6-8 months after grafting. After vitro fertilization of oocyte retrieved, four early embryos were derived. Following embryo transfer, one patient became pregnant and delivered a healthy baby boy, with three other embryos under cryopreservation.IVA technology can effectively activate residual follicles in some patients with POI and allow them to conceive their own genetic offspring. IVA may also be useful for treating patients with ovarian dysfunction including aging women and cancer survivors.
View details for PubMedID 27571179
Premature ovarian failure (POF) is diagnosed by amenorrhea before 40 years of age. Owing to exhaustion of follicles in POF ovaries, egg donation is the only option. Although menstrual cycles cease in POF patients, some of them still contain residual dormant follicles in ovaries. Recently, we developed a new infertility treatment and named it as in-vitro activation (IVA), which enables POF patients to conceive using their own eggs by activation of residual dormant follicles. Here, we summarize data showing the potential of IVA as a new infertility treatment for POF patients.Transgenic mouse studies revealed that the stimulation of phosphatidylinositol-3-kinase-AKT-forkhead box O3 pathway activated dormant primordial follicles. In murine and human ovaries, the phosphatase and tensin homolog inhibitors and phosphatidylinositol-3-kinase activators were demonstrated to activate dormant primordial follicles in in-vitro cultures. Subsequent studies showed that ovarian fragmentation suppressed Hippo signaling pathway, leading to ovarian follicle growth. Combining these two methods in an IVA approach followed by ovarian tissue autotransplantation, successful follicle growth, and pregnancies were reported in POF patients. Currently, two healthy babies were delivered, together with two additional pregnancies.IVA treatment is a potential infertility therapy for POF patients who have residual follicles.
View details for DOI 10.1097/GCO.0000000000000268
View details for Web of Science ID 000375150600013
View details for PubMedID 27022685
Apela (APJ early endogenous ligand, also known as elabela or toddler) is a recently discovered peptide hormone. Based on genetic studies in zebrafish, apela was found to be important for endoderm differentiation and heart development during embryogenesis. Although common phenotypes of apela and APJ-null zebrafish during embryonic development suggested that apela interacts with the APJ receptor, kinetics of apela binding to APJ and intracellular signaling pathways for apela remain unknown. The role of apela in adults is also uncertain. Using a chimeric apela ligand, we showed direct binding of apela to APJ with high affinity (Kd = 0.51 nm) and the ability of apelin, the known peptide ligand for APJ, to compete for apela binding. Apela, similar to apelin, acts through the inhibitory G protein pathway by inhibiting forskolin-stimulated cAMP production and by inducing ERK1/2 phosphorylation. In adult rats, apela is expressed exclusively in the kidney, unlike the wide tissue distribution of apelin. In vivo studies demonstrated the ability of apela to regulate fluid homeostasis by increasing diuresis and water intake. Dose-response studies further indicated that apela induces 2- and 5-fold higher maximal responses than apelin in ERK1/2 phosphorylation and diuresis/water intake, respectively. After designing an apela antagonist, we further demonstrated the role of endogenous ligand(s) in regulating APJ-mediated fluid homeostasis. Our results identified apela as a potent peptide hormone capable of regulating fluid homeostasis in adult kidney through coupling to the APJ-mediated Gi signaling pathway.
View details for DOI 10.1074/jbc.M115.648238
View details for Web of Science ID 000358512100001
View details for PubMedCentralID PMC4513087
Hippo signaling pathway consists of conserved serine/threonine kinases to maintain optimal organ sizes. Studies have demonstrated that fragmentation of murine ovaries increases actin polymerization and disrupts Hippo signaling, leading to nuclear translocation of Hippo signaling effector Yes-associated protein (YAP) in ovarian follicles and follicle growth. For patients with polycystic ovarian syndrome showing follicle arrest, ovarian wedge resection and laser drilling promote follicle growth. Because these damaging procedures likely involve actin polymerization, we tested whether actin polymerization-promoting drugs could promote YAP translocation and stimulate follicle growth. Treatment of murine ovaries with μM Jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, increased the conversion of globular actin to the filamentous form, followed by increased nuclear YAP and expression of downstream connective tissue growth factor (CCN2). After short-term treatments with JASP or S1P, in vitro cultured and in vivo grafted ovaries showed follicle growth. Furthermore, induction of constitutively active YAP in ovarian grafts of transgenic mice enhanced follicle development, whereas treatment of human ovarian cortices with JASP or S1P increased CCN2 expression. Thus, JASP and S1P stimulate follicle growth and are potential therapeutic agents for treating polycystic ovarian syndrome and other ovarian disorders.
View details for DOI 10.1096/fj.14-267856
View details for Web of Science ID 000355209500019
View details for PubMedID 25690654
View details for PubMedCentralID PMC4447223
Although hormonal regulation of ovarian follicle development has been extensively investigated, most studies concentrate on the development of early antral follicles to the preovulatory stage, leading to the successful use of exogenous FSH for infertility treatment. Accumulating data indicate that preantral follicles are under stringent regulation by FSH and local intraovarian factors, thus providing the possibility to develop new therapeutic approaches. Granulosa cell-derived C-type natriuretic factor not only suppresses the final maturation of oocytes to undergo germinal vesicle breakdown before ovulation but also promotes preantral and antral follicle growth. In addition, several oocyte- and granulosa cell-derived factors stimulate preantral follicle growth by acting through wingless, receptor tyrosine kinase, receptor serine kinase, and other signaling pathways. In contrast, the ovarian Hippo signaling pathway constrains follicle growth and disruption of Hippo signaling promotes the secretion of downstream CCN growth factors capable of promoting follicle growth. Although the exact hormonal factors involved in primordial follicle activation has yet to be elucidated, the protein kinase B (AKT) and mammalian target of rapamycin signaling pathways are important for the activation of dormant primordial follicles. Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, promote the growth of preantral follicles in patients with primary ovarian insufficiency, leading to a new infertility intervention for such patients. Elucidation of intraovarian mechanisms underlying early folliculogenesis may allow the development of novel therapeutic strategies for patients diagnosed with primary ovarian insufficiency, polycystic ovary syndrome, and poor ovarian response to FSH stimulation, as well as for infertile women of advanced reproductive age.
View details for DOI 10.1210/er.2014-1020
View details for PubMedID 25202833
View details for PubMedCentralID PMC4309737
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and mTOR signaling is important in regulating cell growth and proliferation. Recent studies using oocyte- and granulosa cell-specific deletion of mTOR inhibitor genes TSC1 or TSC2 demonstrated the important role of mTOR signaling in the promotion of ovarian follicle development. We now report that treatment of ovaries from juvenile mice with an mTOR activator MHY1485 stimulated mTOR, S6K1 and rpS6 phosphorylation. Culturing ovaries for 4 days with MHY1485 increased ovarian explant weights and follicle development. In vivo studies further demonstrated that pre-incubation of these ovaries with MHY1485 for 2 days, followed by allo-grafting into kidney capsules of adult ovariectomized hosts for 5 days, led to marked increases in graft weights and promotion of follicle development. Mature oocytes derived from MHY1485-activated ovarian grafts could be successfully fertilized, leading the delivery of healthy pups. We further treated ovaries with the mTOR activator together with AKT activators (PTEN inhibitor and phosphoinositol-3-kinase stimulator) before grafting and found additive enhancement of follicle growth. Our studies demonstrate the ability of an mTOR activator in promoting follicle growth, leading to a potential strategy to stimulate preantral follicle growth in infertile patients.
View details for DOI 10.1371/journal.pone.0117769
View details for PubMedID 25710488
View details for PubMedCentralID PMC4340052
Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.
View details for DOI 10.1371/journal.pone.0106804
View details for Web of Science ID 000341271500089
View details for PubMedID 25188337
View details for PubMedCentralID PMC4154783
Using accumulating SNP (Single-Nucleotide Polymorphism) data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS), a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L(6)-NPS) causing non-synonymous substitution on the 6(th) position (V to L) of the NPS mature peptide region. L(6)-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ~25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L(6)-NPS exhibits a significant lower bioactivity than the wild type NPS, with ~20-fold higher EC50 values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6(th) position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.
View details for DOI 10.1371/journal.pone.0083009
View details for Web of Science ID 000329117900017
View details for PubMedID 24386135
View details for PubMedCentralID PMC3873911
Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.
View details for DOI 10.1073/pnas.1312830110
View details for Web of Science ID 000325943300068
View details for PubMedID 24082083
View details for PubMedCentralID PMC3808580
Hippo pathway controls the organ size by modulating cell proliferation and apoptosis. However, the upstream regulation of hippo signaling by actin cytoskeleton is not clear. To elucidate the role of actin as an upstream regulator of Hippo signaling, the levels of F (filamentous)-actin in cells were elevated using jasplakinolide, an actin-stabilizing drug. Induction of F-actin formation in HeLa cells resulted in decreased phosphorylation of YAP, a key effector molecule for Hippo signaling. The activated YAP is localized to the cell nucleus and YAP increase was associated with increased expression of downstream CCN growth factors CCN1/CYR61 and CCN2/CTGF. The effect of the actin-stabilizing drug was blocked when YAP levels were suppressed in YAP "knock-down" cells. In summary, using an actin-stabilizing drug we show that actin cytoskeleton is one of the upstream regulators of Hippo signaling capable of activating YAP and increasing its downstream CCN growth factors.
View details for DOI 10.1371/journal.pone.0073763
View details for Web of Science ID 000326734500048
View details for PubMedID 24040060
View details for PubMedCentralID PMC3770699
R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.-Cheng, Y., Kawamura, K., Takae, S., Deguchi, M., Yang, Q., Kuo, C., Hsueh, A. J. W. Oocyte-derived R-spondin2 promotes ovarian follicle development.
View details for DOI 10.1096/fj.12-223412
View details for PubMedID 23407710
View details for PubMedCentralID PMC3659360
Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.
View details for DOI 10.1242/jcs.123471
View details for Web of Science ID 000319561700017
View details for PubMedID 23444378
View details for PubMedCentralID PMC3666258
Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.
View details for DOI 10.1111/j.1365-2826.2012.02381.x
View details for Web of Science ID 000314072400011
View details for PubMedID 22963497
Alternative splicing of genes generates novel mRNAs, leading to the evolution of new functional proteins. Cholecystokinin (CCK) induces the release of pancreatic enzymes and the contraction of the gallbladder to promote the digestion of fat and proteins. CCK activates two G-protein-coupled receptors, CCKA and CCKB. Here, we showed that a CCKsv (splicing variant), originated de novo during Catarrhini evolution by including a portion of intronic sequence of the CCK gene, encodes novel C-terminal peptide sequence followed by a new poly-adenylation signal. CCKsv is expressed in many human tissues and likely a secreted peptide retaining the original signal peptide and the N-terminal proteolytic processing signal, together with novel C-terminal sequences. Although CCKsv cannot activate CCK receptors, it partially inhibits the CRE- or SRF-driven reporter activities stimulated by wide type CCK-8 mediated by both CCK receptors. Co-treatment with CCKsv also partially antagonizes Ewing tumor cell growth stimulated by CCK-8. Our study provides an example of new peptide hormone antagonist evolution in primates.
View details for DOI 10.1371/journal.pone.0064610
View details for PubMedID 23724068
View details for PubMedCentralID PMC3665846
Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.
View details for DOI 10.1371/journal.pone.0049328
View details for Web of Science ID 000311234000064
View details for PubMedID 23152897
View details for PubMedCentralID PMC3495911
C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.
View details for DOI 10.1210/me.2012-1027
View details for Web of Science ID 000305962500008
View details for PubMedID 22595960
View details for PubMedCentralID PMC3385790
Ovarian Kaleidoscope database (OKdb) is an online, searchable, public database containing text-based and DNA microarray data to facilitate research by ovarian researchers. Using key words and predetermined categories, users can search ovarian gene information based on gene function, cell type of expression, cellular localization, hormonal regulation, mutant phenotypes, chromosomal location, ligand-receptor relationship, and other criteria, either alone or in combination. For individual genes, users can access more than 10 extensive DNA microarray datasets to interrogate gene expression patterns in a development-specific and cell type-specific manner. All ligand and receptor genes expressed in the ovary are matched to facilitate investigation of paracrine/autocrine signaling. More than 3500 ovarian genes in the database are matched to 185 gene pathways in the Kyoto Encyclopedia of Genes and Genomes to allow for elucidation of gene interactions and relationships. In addition to >400 genes with infertility or subfertility phenotypes when mutated in mice or humans, the OKdb also lists ~50 and ~40 genes associated with polycystic ovarian syndrome and primary ovarian insufficiency, respectively. The expanding OKdb is updated weekly and allows submission of new genes by ovarian researchers to allow instant access to DNA microarray datasets for newly submitted genes. The present database is a virtual community for ovarian researchers and allows users to instantaneously provide their comments for individual gene pages based on an automated Web-discussion system. In the coming years, we will continue to add new features to serve the ovarian research community.
View details for DOI 10.1095/biolreprod.112.099127
View details for Web of Science ID 000306548000023
View details for PubMedID 22441797
View details for PubMedCentralID PMC3386152
In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.
View details for DOI 10.1210/me.2011-1187
View details for Web of Science ID 000300004700011
View details for PubMedID 22207716
View details for PubMedCentralID PMC3275165
It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug.
View details for DOI 10.1371/journal.pone.0030649
View details for Web of Science ID 000301639600041
View details for PubMedID 22292006
View details for PubMedCentralID PMC3265514
In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. Recently, C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC) was found to suppress mouse oocyte maturation by promoting cyclic guanosine 5'-monophospate (cGMP) production in cumulus cells. However, regulation of NPPC/CNP expression during the pre-ovulatory period and their regulation by the LH surge have not been investigated.Based on genome-wide analysis of DNA microarray data sets using samples from periovulatory ovaries, we found increases in NPPC transcripts in granulosa cells during pre-ovulatory follicle growth in mice and a rapid decline induced by the pre-ovulatory LH/hCG stimulation. Treatment of pre-ovulatory animals with hCG decreased ovarian CNP content. In isolated ovarian cells, NPPC mRNA was predominantly expressed in mural granulosa cells exhibiting similar regulation following gonadotrophin treatment. In cultured mouse pre-ovulatory follicles, meiosis resumption in oocytes by hCG treatment was accompanied by decreases in NPPC transcript levels. In cultured mouse cumulus cell-oocyte complexes, CNP treatment inhibited the resumption of meiosis with increases in cGMP levels in both cumulus cells and oocytes. In human ovaries, CNP levels in ovarian follicular fluid were also decreased following treatment of patients with an ovulatory dose of hCG.Our findings demonstrate gonadotrophins regulation of NPPC/CNP expression in mouse and human ovaries and confirm the role of CNP as a potent paracrine oocyte maturation inhibitor.
View details for DOI 10.1093/humrep/der282
View details for Web of Science ID 000296106600024
View details for PubMedID 21865234
Development of ovarian follicles is regulated by pituitary-derived gonadotropins together with local ovarian paracrine factors. Based on DNA microarray data, we performed RT-PCR and immunostaining to demonstrate the expression of interleukin 7 transcripts in oocytes of preantral, antral, and preovulatory follicles in rats. We also found the expression of interleukin 7 receptor and the coreceptor interleukin 2 receptor gamma in granulosa cells, cumulus cells, and preovulatory oocytes. In cultured rat granulosa cells obtained from early antral and preovulatory follicles, treatment with interleukin 7 stimulated the phosphorylation of AKT, glycogen synthase kinase (GSK3B), and STAT5 proteins in a time- and dose-dependent manner. Furthermore, measurement of mitochondrial reductase activity indicated that treatment with interleukin 7, similar to gonadotropins, increased the number of viable granulosa cells during a 24-h culture period. Furthermore, monitoring of the activities of apoptotic enzymes (caspase 3/7) indicated that treatment with interleukin 7 suppressed apoptosis of cultured granulosa cells from both antral and preovulatory follicles following serum withdrawal. The apoptosis-suppressing actions of interleukin 7 were blocked by an inhibitor of the phosphoinositol-3-kinase (PIK3)/AKT pathway. Furthermore, treatment of cultured preovulatory follicles with interleukin 7, like treatment with human chorionic gonadotropin, induced germinal vesicle breakdown of oocytes. The stimulatory effect of interleukin 7 was also blocked by inhibitors of the PIK3/AKT pathway. The present findings suggest that oocyte-derived interleukin 7 could act on neighboring granulosa cells as a survival factor and promote the nuclear maturation of preovulatory oocytes through activation of the PIK3/AKT pathway.
View details for DOI 10.1095/biolreprod.110.086504
View details for Web of Science ID 000288596900012
View details for PubMedID 21178173
In mammalian ovaries, diverse paracrine factors have been identified to mediate or modulate LH-induced changes during ovulation. Due to the difficulty in obtaining non-stimulated granulosa cells during IVF, little is known about the LH-induced paracrine factors in the human ovary. Based on earlier studies using murine ovarian cells showing the paracrine roles of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in promoting oocyte maturation, we investigated the expression of these ligands in human granulosa cells and their regulation of human oocyte development.Non-stimulated granulosa cells were obtained from non-stimulated IVM (in vitro maturation) patients after oocyte retrieval. Women undergoing non-stimulated IVM treatment at a mean age of 30.8 ± 1.3 (n = 10) were recruited for this study. Immature oocytes and granulosa cells were collected from IVF patients undergoing gonadotrophin stimulation and ICSI. Immunocytochemical analyses of granulosa cells were carried out to investigate expression profiles of BDNF and GDNF, together with real-time RT-PCR to analyze the gonadotrophin regulation of BDNF and GDNF transcript levels. In addition, immature oocytes were cultured to analyze the regulation of oocyte maturation by BDNF and GDNF.BDNF and GDNF were found to be expressed in non-stimulated granulosa cells. After gonadotrophin (FSH and/or hCG) treatment, transcripts levels for BDNF and GDNF were significantly increased (P < 0.05). In cultured immature oocytes, treatment with BDNF or GDNF promoted total yields of metaphase II oocytes.These findings demonstrate that FSH and hCG treatments augment the expression of BDNF and GDNF by granulosa cells and that these granulosa-cell-derived factors are candidate paracrine factors capable of promoting oocyte maturation.
View details for DOI 10.1093/humrep/deq390
View details for Web of Science ID 000287252800026
View details for PubMedID 21227937
Although multiple follicles are present in mammalian ovaries, most of them remain dormant for years or decades. During reproductive life, some follicles are activated for development. Genetically modified mouse models with oocyte-specific deletion of genes in the PTEN-PI3K-Akt-Foxo3 pathway exhibited premature activation of all dormant follicles. Using an inhibitor of the Phosphatase with TENsin homology deleted in chromosome 10 (PTEN) phosphatase and a PI3K activating peptide, we found that short-term treatment of neonatal mouse ovaries increased nuclear exclusion of Foxo3 in primordial oocytes. After transplantation under kidney capsules of ovariectomized hosts, treated follicles developed to the preovulatory stage with mature eggs displaying normal epigenetic changes of imprinted genes. After in vitro fertilization and embryo transfer, healthy progeny with proven fertility were delivered. Human ovarian cortical fragments from cancer patients were also treated with the PTEN inhibitor. After xeno-transplantation to immune-deficient mice for 6 months, primordial follicles developed to the preovulatory stage with oocytes capable of undergoing nuclear maturation. Major differences between male and female mammals are unlimited number of sperm and paucity of mature oocytes. Thus, short-term in vitro activation of dormant ovarian follicles after stimulation of the PI3K-Akt pathway allows the generation of a large supply of mature female germ cells for future treatment of infertile women with a diminishing ovarian reserve and for cancer patients with cryo-preserved ovaries. Generation of a large number of human oocytes also facilitates future derivation of embryonic stem cells for regenerative medicine.
View details for DOI 10.1073/pnas.1001198107
View details for Web of Science ID 000278246000066
View details for PubMedID 20479243
View details for PubMedCentralID PMC2890455
Spontaneous premature ovarian failure (POF) occurs in 1% of women and has major implications for their fertility and health. Besides X chromosomal aberrations and fragile X premutations, no common genetic risk factor has so far been discovered in POF. Using high-density single nucleotide polymorphism (SNP) arrays, we set out to identify new genetic variants involved in this condition.A genome-wide association study involving 309 158 SNPs was performed in 99 unrelated idiopathic Caucasian POF patients and 235 unrelated Caucasian female controls. A replication study on the most significant finding was performed. We specifically focused on chromosomal areas and candidate genes previously implicated in POF.Suggestive genome-wide significant association was observed for rs246246 (allele frequency P = 6.0 x 10(-7)) which mapped to an intron of ADAMTS19, a gene known to be up-regulated in the female mouse gonads during sexual differentiation. However, replication in an independent Dutch cohort (60 POF patients and 90 controls) could not confirm a clear association (P = 4.1 x 10(-5) in a joint analysis). We did not observe strong evidence for any of 74 selected POF candidate genes or linkage regions being associated with idiopathic POF in Caucasian females, although suggestive association (P < 0.005) was observed for SNPs that mapped in BDNF, CXCL12, LHR, USP9X and TAF4B.We observed a possible association between POF and a SNP in a biologically plausible candidate gene. Although limited by sample size, this proof-of-principle study's findings reveal ADAMTS19 as a possible candidate gene for POF and thus a larger follow-up study is warranted.
View details for DOI 10.1093/humrep/dep197
View details for Web of Science ID 000269001600038
View details for PubMedID 19508998
Development of early embryos is regulated by autocrine/paracrine factors. Analyzing the expression of polypeptide ligand-receptor pairs using DNA microarray datasets, we identified transcripts for artemin, a member of the GDNF (glial cell line-derived neurotrophic factor) family, its receptor GFRA3 (GDNF family receptor-alpha 3) and coreceptor RET. Here we report an autocrine/paracrine role of the artemin-GFRA3 signaling system in regulating early embryonic development and apoptosis. Possible involvement of the MAP kinase signaling pathway was also demonstrated. The genome-wide survey of ligand-receptor pairs and early embryo cultures provided a better understanding of autocrine/paracrine embryonic factors important for optimal blastocyst development.
View details for DOI 10.1016/j.febslet.2009.06.050
View details for Web of Science ID 000268563500007
View details for PubMedID 19580811
The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarian Ihh and Dhh transcripts decreased 4-24 h after hCG versus 0-48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mouse Ptch1 and Smo transcripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system with Ihh mRNA abundance in granulosa cells was greater, whereas Smo and Ptch1 mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance of Gli1 and Ptch1 mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreased Ihh mRNA abundance in bovine granulosa cells. The expression and regulation of Ihh transcripts in granulosa cells and Ptch1 mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.
View details for DOI 10.1530/REP-08-0317
View details for Web of Science ID 000268344700014
View details for PubMedID 19433502
Shortly after stimulation by the preovulatory surge of luteinizing hormone (LH), oocytes arrested at the late prophase I resume meiosis characterized by germinal vesicle breakdown (GVBD), chromosome condensation, and extrusion of the first polar body in preparation for fertilization and early embryonic development. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation. Thus, factors released by granulosa or theca cells expect to convey the LH stimuli to oocytes. To identify candidate ligand-receptor pairs potentially involved in the process of oocyte maturation, we performed DNA microarray analyses of ovarian transcripts in mice and identified Kit ligand (Kitl) as an ovarian factor stimulated by the LH/hCG surge. The purpose of this study is to investigate the roles of KITL in the nuclear and cytoplasmic maturation of preovulatory mouse oocytes.The levels of Kitl and c-kit transcripts in mouse ovaries and isolated ovarian cells were determined by real-time RT-PCR, while expression of KITL protein was examined by immunohistochemistry. Follicle culture, cumulus-oocyte complexes (COC) and denuded oocytes culture were used to evaluate the effect of KITL on mouse oocyte nuclear maturation. To assess the effect of KITL treatment on the cytoplasmic maturation of preovulatory oocytes, we performed in vitro maturation of oocytes followed by in vitro fertilization.Major increase of Kitl transcripts in granulosa cells and mouse ovaries, and predominant expression of c-kit in preovulatory oocytes were identified by real-time RT-PCR. Predominant expression of KITL protein was found in granulosa cells of preovulatory and small antral follicles at 4 h after hCG treatment. In vitro cultures demonstrated that treatment with KITL enhanced first polar body extrusion in a dose-dependent manner. Moreover, treatment of COC with KITL enhanced first polar body extrusion with increase in cyclin B1 synthesis which is important for the progression of meiotic maturation after GVBD. In contrast, treatment of cultured preovulatory follicles with KITL did not affect GVBD and KITL has no effect on cytoplasmic maturation of preovulatory oocytes.Our findings suggest potential paracrine roles of KITL in the nuclear maturation of preovulatory oocytes by promoting first polar body extrusion.
View details for DOI 10.1186/1477-7827-7-26
View details for Web of Science ID 000266491000001
View details for PubMedID 19341483
View details for PubMedCentralID PMC2676294
Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus-oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge.
View details for DOI 10.1016/j.ydbio.2008.11.033
View details for Web of Science ID 000263706200007
View details for PubMedID 19111534
Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide.
View details for DOI 10.1074/jbc.M804784200
View details for Web of Science ID 000260760800077
View details for PubMedID 18753129
View details for PubMedCentralID PMC2581552
The proprotein convertase subtilisin/kexin (PCSKs), a family of subtilisin-like proteases, is the processing enzymes for the activation of many hormone precursors. The present study was designed to identify the PCSK isoform expressed in the ovary and to examine its expression in gonadotropin-stimulated rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of Pcsk5 messenger RNA (mRNA) during development with the highest levels at 21 days of age. Treatment of immature rats with PMSG further increased ovarian Pcsk5 expression, and in situ hybridization analysis revealed the localization of Pcsk5 mRNA in theca-interstitial cells of follicles in different sizes. Interestingly, treatment of PMSG-primed rats with hCG resulted in a transient stimulation of ovarian Pcsk5 mRNA levels within 3-6 h. In addition to theca-interstitial cells, hCG treatment induced the expression of Pcsk5 in granulosa cells of preovulatory follicles. Pcsk1, 2 and 4 mRNAs were not detected whereas Pcsk7 mRNA was slightly expressed. Injection of a progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane at 1h before hCG treatment inhibited hCG-induced Pcsk5 mRNA levels. Treatment with LH stimulated both Pcsk5 mRNA and protein levels in preovulatory follicles cultured in vitro. In addition, forskolin but not TPA stimulated Pcsk5 mRNA levels. RNase protection assay revealed that the soluble Pcsk5A variant was the predominant form stimulated by gonadotropins in the ovary. Finally, the predicted proprotein substrates cleaved by PCSK5 were analyzed in preovulatory follicles using regular expressions. The present study demonstrates PCSK5A as the gonadotropin-regulated PCSK isoform in the ovary, and its possible contribution to ovulation by processing pro-TGFbeta and matrix metalloproteinase family.
View details for DOI 10.1016/j.mce.2008.04.006
View details for Web of Science ID 000257841700005
View details for PubMedID 18502031
Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.
View details for DOI 10.1210/me.2007-0569
View details for Web of Science ID 000256307800016
View details for PubMedID 18337590
View details for PubMedCentralID PMC5419543
Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3beta-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.
View details for DOI 10.1210/en.2007-1662
View details for Web of Science ID 000256053100015
View details for PubMedID 18292196
View details for PubMedCentralID PMC2408816
Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus-oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos.
View details for DOI 10.1016/j.ydbio.2007.12.029
View details for Web of Science ID 000253750300015
View details for PubMedID 18234170
Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.
View details for DOI 10.1095/biolreprod.107.063446
View details for Web of Science ID 000252525300005
View details for PubMedID 17959852
Hormonal factors secreted by embryos and reproductive tracts are important for successful development of preimplantation embryos. We found expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) transcripts at its highest levels in the blastocyst stages. The transcripts for their receptor, TrkB, were detectable throughout the early embryonic stages with an increase after the early blastocyst stage. Both BDNF and TrkB are expressed in trophectoderm cells, whereas ligand-binding studies indicated specific binding of BDNF to trophectoderm cells. Furthermore, BDNF and NT-4/5 were produced in pregnant oviducts and uteri. Treatment with BDNF promoted the development of two-cell-stage embryos into blastocysts showing increased proliferation and decreased apoptosis. The effects of BDNF were blocked by the TrkB ectodomain or a Trk receptor inhibitor, K252a. Studies using specific inhibitors demonstrated the roles of the PI3K, but not the ERK, pathway in mediating BDNF actions. Under high-density embryo cultures, treatment with the TrkB ectodomain or K252a alone also inhibited embryonic development and survival, suggesting potential autocrine actions of BDNF produced by the embryo. In vivo experiments further demonstrated that K252a treatment suppressed early embryo development by inhibiting blastocyst cell numbers, and increasing blastocyst apoptosis. Our findings suggested that BDNF signaling plays important paracrine roles during blastocyst development by promoting the development of preimplantation embryos.
View details for DOI 10.1016/j.ydbio.2007.08.026
View details for Web of Science ID 000250703700012
View details for PubMedID 17880937
Sequencing of genomes from diverse organisms facilitates studies on the repertoire of genes involved in intercellular signaling. Extending previous efforts to annotate most human plasma membrane receptors in the Human Plasma Membrane Receptome database, we matched cognate ligands with individual receptors by surveying the published literature. In the updated online database we called "liganded receptome," users can search for individual ligands or receptors to reveal their pairing partners and browse through receptor or ligand families to identify relationships between ligands and receptors in their respective families. Because local signaling systems are prevalent in diverse normal and diseased tissues, we used the liganded receptome knowledgebase to interrogate DNA microarray datasets for genome-wide analyses of potential paracrine/autocrine signaling systems. In addition to viewing ligand-receptor coexpression based on precomputed DNA microarray data, users can submit their own microarray data to perform online genome-wide searches for putative paracrine/autocrine signaling systems. Investigation of transcriptome data based on liganded receptome allows the discovery of paracrine/autocrine signaling for known ligand-receptor pairs in previously uncharacterized tissues or developmental stages. The present annotation of ligand-receptor pairs also identifies orphan receptors and ligands without known interacting partners in select families. Because hormonal ligands within the same family usually interact with paralogous receptors, this genomic approach could also facilitate matching of orphan receptors and ligands. The liganded receptome is accessible at http://receptome.stanford.edu.
View details for DOI 10.1210/me.2007-0087
View details for Web of Science ID 000248324600020
View details for PubMedID 17550980
The relaxin and insulin-like peptide 3 receptors, LGR7 and LGR8, respectively, are unique members of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, because they possess an N-terminal motif with homology to the low density lipoprotein class A (LDLa) modules. By characterizing several LGR7 and LGR8 splice variants, we have revealed that the LDLa module directs ligand-activated cAMP signaling. The LGR8-short variant encodes an LGR8 receptor lacking the LDLa module, whereas LGR7-truncate, LGR7-truncate-2, and LGR7-truncate-3 all encode truncated secreted proteins retaining the LGR7 LDLa module. LGR8-short and an engineered LGR7 variant missing its LDLa module, LGR7-short, bound to their respective ligands with high affinity but lost their ability to signal via stimulation of intracellular cAMP accumulation. Conversely, secreted LGR7-truncate protein with the LDLa module was able to block relaxin-induced LGR7 cAMP signaling and did so without compromising the ability of LGR7 to bind to relaxin or be expressed on the cell membrane. Although the LDLa module of LGR7 was N-glycosylated at position Asn-14, an LGR7 N14Q mutant retained relaxin binding affinity and cAMP signaling, implying that glycosylation is not essential for optimal LDLa function. Using real-time PCR, the expression of mouse LGR7-truncate was detected to be high in, and specific to, the uterus of pregnant mice. The differential expression and evolutionary conservation of LGR7-truncate further suggests that it may also play an important role in vivo. This study highlights the essential role of the LDLa module in LGR7 and LGR8 function and introduces a novel model of GPCR regulation.
View details for DOI 10.1074/jbc.M602728200
View details for Web of Science ID 000241933700027
View details for PubMedID 16963451
In addition to gonadotropins, many ovarian paracrine factors are crucial for optimal follicle rupture, oocyte maturation, and luteinization. Based on DNA microarray analyses, we found that transcripts for the fibroblast growth factor-inducible-14 (Fn14) receptor are increased after LH/human chorionic gonadotropin (hCG) treatment of gonadotropin-primed immature mice or rats. Fn14 is the cognate receptor for TNF-related weak inducer of apoptosis (TWEAK), a TNF superfamily member. TWEAK transcripts also were detected in the ovary; however, their levels were not regulated by gonadotropins. In situ hybridization analyses indicated that the Fn14 receptor is expressed in the granulosa and cumulus cells of preovulatory follicles and, to a lesser extent, in theca cells. In contrast, in situ hybridization analyses revealed that TWEAK is primarily expressed in theca cells. In cultured granulosa cells pretreated with hCG to induce Fn14 receptor expression, treatment with TWEAK suppressed progesterone synthesis without accompanying changes in cAMP production. Furthermore, intrabursal injection of TWEAK suppressed ovarian progesterone content in gonadotropin-primed rats. In contrast, preovulatory follicles cultured in the presence of the Fn14 decoy, a recombinant protein containing the ligand-binding domain of Fn14, led to increases in progesterone production, presumably by antagonizing the actions of endogenous TWEAK. Likewise, ip injection of the Fn14 decoy enhanced serum progesterone levels with accompanying increases in transcript levels for several key steroidogenic enzymes. The present findings demonstrate a suppressive role of the TWEAK/Fn14 signaling system in the ovary. Following gonadotropin induction of ovulation, Fn14 is induced and could protect preovulatory follicles from excessive luteinization.
View details for DOI 10.1210/me.2006-0028
View details for Web of Science ID 000240785100022
View details for PubMedID 16762976
Recent advances in genomic sequencing allow a new paradigm in hormonal research, and a comparative genomic approach facilitates the identification of receptors and signalling mechanisms for orphan ligands of the transforming growth factor beta (TGFbeta) superfamily. Instead of purifying growth differentiation factor 9 (GDF9) receptor proteins for identification, we hypothesized that GDF9, like other ligands in the TGFbeta family, activates type II and type I serine/threonine kinase receptors. Because searches of the human genome for genes with sequence homology to known serine/threonine kinase receptors failed to reveal uncharacterized receptor genes, GDF9 likely interacts with the known type II and type I activin receptor-like kinase (ALK) receptors in granulosa cells. We found that co-treatment with the bone morphogenetic protein (BMP) type II receptor (BMPRII) ectodomain blocks GDF9 activity. Likewise, in a GDF9-non-responsive cell line, overexpression of ALK5, but none of the other six type I receptors, conferred GDF9 responsiveness. The roles of BMPRII and ALK5 as receptors for GDF9 were validated in granulosa cells using gene "knock-down" approaches. Furthermore, we demonstrated the roles of BMPRII, ALK3 and ALK6 as the receptors for the orphan ligands GDF6, GDF7 and BMP10. Thus, evolutionary tracing of polypeptide ligands, receptors and downstream signalling molecules in their respective 'subgenomes' facilitates a new approach for hormonal research.
View details for DOI 10.1093/humupd/dml014
View details for Web of Science ID 000238537800005
View details for PubMedID 16603567
In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.
View details for DOI 10.1677/joe.1.06503
View details for Web of Science ID 000237730900014
View details for PubMedID 16648300
Recent completion of the sequencing of genomes from several mammals, teleosts and invertebrates has shown that G protein-coupled receptors (GPCRs) are one of the conserved groups of cell surface receptors with an ancient origin. GPCRs play important roles in diverse physiological functions and are the most important targets for pharmaceutical discoveries. Recent work based on the search for gene with structural similarity to LH, FSH and thyroid-stimulating hormone (TSH) receptors in diverse genomes has led to the identification of a group of GPCRs called Leucine-rich repeat-containing, G protein-coupled Receptor (LGR). We present the genomic analyses of the evolution of LGR genes in the literature.
View details for PubMedID 16642723
Recent advances in comparative genomics allow a new paradigm for hormonal research. At the centennial of the first use of the term hormone by Ernest Starling, we reflected on the changing approaches in elucidating hormonal signaling mechanisms and highlighted the inadequacy of the term endocrinology, implying remote activation, to describe the diverse modes of hormone actions. Several examples were presented to underscore the power of comparative genomics in the identification of new polypeptide hormones, receptors, and signaling pathways. We propose the use of the term hormonology to more accurately reflect the expanding boundaries of the discipline.
View details for DOI 10.1677/joe.1.06372
View details for Web of Science ID 000234210200003
View details for PubMedID 16423812
Ghrelin, a circulating appetite-inducing hormone, is derived from a prohormone by posttranslational processing. On the basis of the bioinformatic prediction that another peptide also derived from proghrelin exists, we isolated a hormone from rat stomach and named it obestatin-a contraction of obese, from the Latin "obedere," meaning to devour, and "statin," denoting suppression. Contrary to the appetite-stimulating effects of ghrelin, treatment of rats with obestatin suppressed food intake, inhibited jejunal contraction, and decreased body-weight gain. Obestatin bound to the orphan G protein-coupled receptor GPR39. Thus, two peptide hormones with opposing action in weight regulation are derived from the same ghrelin gene. After differential modification, these hormones activate distinct receptors.
View details for DOI 10.1126/science.1117255
View details for PubMedID 16284174
There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.
View details for DOI 10.1074/jbc.M504629200
View details for Web of Science ID 000231794800012
View details for PubMedID 16049014
Glycoprotein hormones play important roles in thyroid and gonadal function in vertebrates. The glycoprotein hormone alpha-subunit forms heterodimers with different beta-subunits to activate TSH or gonadotropin (LH and FSH) receptors. Recent genomic analyses allowed the identification of another alpha-subunit, GPA2, and another beta-subunit, GPB5, in human, capable of forming heterodimers to activate TSH receptors. Based on comparative genomic searches, we isolated the fly orthologs for human GPA2 and GPB5, each consisting of 10 cysteine residues likely involved in cystine-knot formation. RT-PCR analyses in Drosophila melanogaster demonstrated the expression of GPA2 and GPB5 at different developmental stages. Immunoblot analyses further showed that fly GPA2 and GPB5 subunit proteins are of approximately 16 kDa, and coexpression of these subunits yielded heterodimers. Purified recombinant fly GPA2/GPB5 heterodimers were found to be glycoproteins with N-linked glycosylated alpha-subunits and nonglycosylated beta-subunits, capable of stimulating cAMP production mediated by fly orphan receptor DLGR1 but not DLGR2. Although the fly GPA2/GPB5 heterodimers did not activate human TSH or gonadotropin receptors, chimeric fly GPA2/human GPB5 heterodimers stimulated human TSH receptors. These findings indicated that fly GPA2/GPB5 is a ligand for DLGR1, thus showing the ancient origin of this glycoprotein hormone-seven transmembrane receptor-G protein signaling system. The fly GPA2 also could form heterodimers with human GPB5 to activate human TSH receptors, indicating the evolutionary conservation of these genes and suggesting that the GPA2 subunit may serve as a scaffold for the beta-subunit to activate downstream G protein-mediated signaling.
View details for DOI 10.1210/en.2005-0317
View details for Web of Science ID 000230427400044
View details for PubMedID 15890769
Optimal development of fertilized eggs into preimplantation embryos is essential for reproduction. Although mammalian oocytes ovulated after luteinizing hormone (LH) stimulation can be fertilized and promoted into early embryos in vitro, little is known about ovarian factors important for the conditioning of eggs for early embryo development. Because LH interacts only with ovarian somatic cells, its potential regulation of oocyte functions is presumably mediated by local paracrine factors. We performed DNA microarray analyses of ovarian transcripts and identified brain-derived neurotrophic factor (BDNF) secreted by granulosa and cumulus cells as an ovarian factor stimulated by the preovulatory LH surge. Ovarian BDNF acts on TrkB receptors expressed exclusively in oocytes to enhance first polar body extrusion of oocytes and to promote the in vitro development of zygotes into preimplantation embryos. Furthermore, in vivo treatment with a Trk receptor inhibitor suppressed first polar body extrusion and the progression of zygotes into blastocysts. Thus, ovarian BDNF is important to nuclear and cytoplasmic maturation of the oocyte, which is essential for successful oocyte development into preimplantation embryos. Treatment with BDNF could condition the cultured oocytes for optimal progression into the totipotent blastocysts.
View details for DOI 10.1073/pnas.0502442102
View details for Web of Science ID 000230191400023
View details for PubMedID 15967989
View details for PubMedCentralID PMC1166611
Intercellular communication relies on signal transduction mediated by extracellular ligands and their receptors. Although the ligand-receptor interaction is usually a two-player event, there are selective examples of one polypeptide ligand interacting with more than one phylogenetically unrelated receptor. Likewise, a few receptors interact with more than one polypeptide ligand, and sometimes with more than one coreceptor, likely through an interlocking of unique protein domains. Phylogenetic analyses suggest that for certain triumvirates, the matching events could have taken place at different evolutionary times. In contrast to a few polypeptide ligands interacting with more than one receptor, we found that many small nonpeptide ligands have been paired with two or more plasma membrane receptors, nuclear receptors, or channels. The observation that many small ligands are paired with more than one receptor type highlights the utilitarian use of a limited number of cellular components during metazoan evolution. These conserved ligands are ubiquitous cell metabolites likely favored by natural selection to establish novel regulatory networks. They likely possess structural features useful for designing agonistic and antagonistic drugs to target diverse receptors.
View details for DOI 10.1210/me.2004-0451
View details for Web of Science ID 000228663900001
View details for PubMedID 15695369
The UCN homologues SCP and SRP bind specifically to the CRFR2 receptor, whereas UCN binds to both CRFR1 and CRFR2. We have previously demonstrated that all three peptides are cardioprotective, and both the Akt and MAPK p42/44 pathways are essential for this effect. Here we tested the hypertrophic effects of these peptides. We examined the effects of the peptides on cell area, protein synthesis, and induction of the natriuretic peptides ANP and BNP. All three peptides were able to increase all the markers of hypertrophy examined, with SCP being the most potent of the three, followed by UCN and SRP last. In addition, we provide a mechanism of action for the three peptides and show that Akt phosphorylation is important for their hypertrophic action, whereas MAPK p42/44 is not involved in this effect.
View details for DOI 10.1016/j.bbrc.2005.01.001
View details for Web of Science ID 000227075700011
View details for PubMedID 15694367
All arthropods periodically molt to replace their exoskeleton (cuticle). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Here we show that bursicon, to our knowledge the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The pburs/burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G protein-coupled receptor DLGR2, leading to the stimulation of cAMP signaling in vitro and tanning in neck-ligated blowflies. Native bursicon from Periplaneta americana is also a heterodimer. In D. melanogaster the levels of pburs, burs, and DLGR2 transcripts are increased before ecdysis, consistent with their role in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species revealed the colocalization of pburs- and burs-immunoreactivity in some of the neurosecretory neurons that also express crustacean cardioactive peptide. Forty-three years after its initial description, the elucidation of the molecular identity of bursicon and the verification of its receptor allow for studies of bursicon actions in regulating cuticle tanning, wing expansion, and as yet unknown functions. Because bursicon subunit genes are homologous to the vertebrate bone morphogenetic protein antagonists, our findings also facilitate investigation on the function of these proteins during vertebrate development.
View details for DOI 10.1073/pnas.0409916102
View details for Web of Science ID 000227232400029
View details for PubMedID 15703293
View details for PubMedCentralID PMC549504
View details for Web of Science ID 000227329101204
In the human ovary, cell growth and differentiation are regulated by members of the TGF-beta superfamily, including growth differentiation factor-9 (GDF9), TGF-beta, and activin. TGF-beta and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-beta, and activin responses. In rodent granulosa cells, TGF-beta and GDF9 signal through the TGF-beta type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase reporter activity. A similar response to Alk5 overexpression was observed for TGF-beta, but not for activin. Adenoviral overexpression of the activin type IB receptor Alk4 in hGL cells specifically potentiated activin signaling, but not GDF9 or TGF-beta signaling. Alk5 overexpression in hGL cells also potentiated the GDF9 response when inhibin B production was used as the read-out. These results indicate that the CAGA-luciferase adenovirus can be used to study Smad3 signaling in primary cultures of human cells, and that adenoviral overexpression of wild-type receptors of the TGF-beta superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-beta, and activin. Furthermore, these studies indicate the involvement of Alk5 in GDF9 signaling in human cells and therefore, along with other recent studies, highlight how a limited number of type I and II receptors cooperate to generate specificity of action within the TGF-beta superfamily.
View details for DOI 10.1210/jc.2004-1288
View details for Web of Science ID 000226230700046
View details for PubMedID 15483083
Stanniocalcin is a glycoprotein hormone important in the maintenance of calcium and phosphate homeostasis in fish. Two related mammalian stanniocalcin genes, STC1 and STC2, were found to be expressed in various tissues as paracrine regulators. We have demonstrated the existence of a second stanniocalcin gene in fish, designated fish STC2, with only 30% identity to fish STC1. However, phylogenetic analysis and comparison of the genomic structure of STC genes in vertebrates indicated that STC1 and STC2 genes were probably derived from a common ancestor gene. Based on the prominent expression of mammalian STC1 in the ovary, we tested STC2 expression in rat ovary and the regulation of STC2 expression by gonadotropins. Treatment of immature rats with pregnant mare serum gonadotropin increased STC2 transcripts, whereas subsequent treatment with human chorionic gonadotropin suppressed STC2 expression. Real-time PCR analyses also demonstrated that STC2 is expressed mainly in thecal layers. In situ hybridization studies also revealed that STC2 is expressed in thecal cell layers of antral and preovulatory follicles after gonadotropin stimulation. To elucidate the physiological functions of STC2, recombinant human and fish STC2 proteins were generated and found to be N-glycosylated homodimers. In cultured granulosa cells, treatment with human or fish STC2 suppressed FSH-induced progesterone, but not estradiol or cAMP, production. The STC2 suppression of progesterone production was associated with the inhibition of FSH-induced CYP11A and 3beta-hydroxysteroid dehydrogenase expression. Thus, STC2 is a functional homodimeric glycoprotein, and thecal cell-derived STC2 could play a paracrine role during follicular development.
View details for DOI 10.1210/en.2004-1197
View details for Web of Science ID 000225766500062
View details for PubMedID 15486227
Because of the coevolution of ligands and their cognate receptors, analysis of human genomic sequences allows prediction of the pairing of these elements. Initially, we identified a group of five human leucine-rich repeat-containing G-protein-coupled receptor (LGR) genes homologous to LH, FSH, and TSH receptors. Based on common phenotypes of INSL3 null mice and transgenic mice with LGR8 gene deletion, we hypothesized that INSL3, relaxin, and related genes are likely ligands for the paralogous LGR7 and LGR8 genes. Matching the relaxin family peptides with these two orphan LGRs led to the finding that relaxin is capable of activating LGR7 and LGR8 through the Gs pathway. In addition, INSL3 and relaxin 3 were found to be specific ligands for LGR8 and LGR7, respectively. Based on the known production of INLS3 by testicular Leydig cells and ovarian theca cells, we demonstrated the expression of the INSL3 receptor LGR8 in oocytes in ovary and in male germ cells in the testis. Furthermore, we found that LH stimulates INSL3 transcripts in ovarian theca and testicular Leydig cells. INSL3, in turn, binds LGR8 expressed in germ cells to initiate the meiotic progression of arrested oocytes in preovulatory follicles in vitro and in vivo and to suppress male germ cell apoptosis in vivo. INSL3 interacts with germ cells to activate the inhibitory G protein, thus leading to decreases in cAMP production. Our data demonstrate the importance of the INSL3-LGR8 paracrine system in mediating gonadotropic actions in both ovary and testis.
View details for Web of Science ID 000231831000002
View details for PubMedID 15956679
The physiological role of an orphan G protein-coupled receptor, LGR5, was investigated by targeted deletion of this seven-transmembrane protein containing a large N-terminal extracellular domain with leucine-rich repeats. LGR5 null mice exhibited 100% neonatal lethality characterized by gastrointestinal tract dilation with air and an absence of milk in the stomach. Gross and histological examination revealed fusion of the tongue to the floor of oral cavity in the mutant newborns and immunostaining of LGR5 expression in the epithelium of the tongue and in the mandible of the wild-type embryos. The observed ankyloglossia phenotype provides a model for understanding the genetic basis of this craniofacial defect in humans and an opportunity to elucidate the physiological role of the LGR5 signaling system during embryonic development.
View details for DOI 10.1128/MCB.24.22.9736-9743.2004
View details for Web of Science ID 000224823300004
View details for PubMedID 15509778
View details for PubMedCentralID PMC525477
Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) belong to the largest mammalian superfamily of proteins with seven-transmembrane domains. LGRs can be divided into three subgroups based on their unique domain arrangement. Although two subgroups have been found to be receptors for glycoprotein hormones and relaxin-related ligands, respectively, the third LGR subgroup, consisting of LGR4-6, are orphan receptors with unknown physiological roles. To elucidate the functions of this subgroup of LGRs, LGR4 null mice were generated using a secretory trap approach to delete the majority of the LGR4 gene after the insertion of a beta-galactosidase reporter gene immediately after exon 1. Tissues expressing LGR4 were analyzed based on histochemical staining of the transgene driven by the endogenous LGR4 promoter. LGR4 was widely expressed in kidney, adrenal gland, stomach, intestine, heart, bone/cartilage, and other tissues. The expression of LGR4 in these tissues was further confirmed by immunohistochemical studies in wild-type animals. Analysis of the viability of 250 newborn animals suggested a skewed inheritance pattern, indicating that only 40% of the expected LGR4 null mice were born. For the LGR4 null mice viable at birth, most of them died within 2 d. Furthermore, the LGR4 null mice showed intrauterine growth retardation as reflected by a 14% decrease in body weight at birth, together with 30% and 40% decreases in kidney and liver weights, respectively. The present findings demonstrate the widespread expression of LGR4, and an essential role of LGR4 for embryonic growth, as well as kidney and liver development. The observed pre- and postnatal lethality of LGR4 null mice illustrates the importance of the LGR4 signaling system for the survival and growth of animals during the perinatal stage.
View details for DOI 10.1210/me.2004-0133
View details for PubMedID 15192078
Stanniocalcin (STC) in fish maintains calcium and phosphate homeostasis, whereas mammalian STC1 shows a diverse tissue expression pattern with ovary exhibiting the highest level. Based on the known expression of STC1 in theca/interstitial cells of the ovary, we generated recombinant N-glycosylated STC1 protein and tested its ability to modulate granulosa cell differentiation. In cultured rat granulosa cells obtained from early antral follicles, treatment with STC1 suppressed FSH-stimulated progesterone biosynthesis with minimal effects on estradiol and cAMP production. In mature granulosa cells, treatment with STC1 also suppressed human chorionic gonadotropin-induced progesterone production. The inhibitory effect of STC1 was accompanied by a pronounced suppression of the CYP11A transcripts and the FSH induction of functional LH receptors. In addition, STC1 was found to act downstream of adenyl cyclases in suppressing progesterone biosynthesis. We also tested the regulation of STC1 gene expression by gonadotropins. Treatment with pregnant mare serum gonadotropin decreased STC1 transcript levels in theca cells of maturing follicles, whereas subsequent treatment with human chorionic gonadotropin led to sustained suppression in the corpora lutea. Using radiolabeled recombinant STC1, receptor assays showed specific STC1 binding with a high affinity to granulosa cells. Because STC1 is expressed in ovarian theca/interstitial cells, the present demonstration of receptor binding and the specific actions of STC1 in granulosa cells suggest the existence of a follicular paracrine system in which theca cell-derived STC1 dampens the gonadotropin stimulation of granulosa cell differentiation. The observed STC1 suppression of progesterone, but not estradiol, production further suggests the potential role of this paracrine hormone as a luteinization inhibitor.
View details for DOI 10.1210/me.2004-0066
View details for Web of Science ID 000222907600018
View details for PubMedID 15131261
Premature ovarian failure in a subgroup of women with blepharophimosis-ptosis-epicanthus inversus type 1 syndrome has been associated with nonsense mutations in the gene encoding a Forkhead transcription factor, Forkhead L2 (FOXL2). However, the exact function of FOXL2 in the ovary is unclear. We investigated the expression of FOXL2 in the mouse ovary during follicular development and maturation by RT-PCR and in situ hybridization. The FOXL2 mRNA is expressed in ovaries throughout development and adulthood and is localized to the undifferentiated granulosa cells in small and medium follicles as well as cumulus cells of preovulatory follicles. FOXL2 belongs to a group of transcription factors capable of interacting with specific DNA sequences in diverse gene promoters. With the presence of multiple putative forkhead DNA consensus sites, the promoter of the human steroidogenic acute regulatory (StAR) gene was used to test for regulation by FOXL2. Cotransfection studies revealed that wild-type FOXL2 represses the activity of the StAR promoter, and the first 95 bp upstream of the transcriptional start site of the StAR gene is sufficient for FOXL2 repression. EMSAs confirmed that FOXL2 interacts directly with this region. Analyses using FOXL2 mutants also demonstrated the importance of the entire alanine/proline-rich carboxyl terminus of FOXL2 for transcriptional repression. Furthermore, these mutations produce a protein with a dominant-negative effect that disables the transcriptional repressor activity of wild-type FOXL2. Dominant-negative mutations of FOXL2 could increase expression of StAR and other follicle differentiation genes in small and medium follicles to accelerate follicle development, resulting in increased initial recruitment of dormant follicles and thus the premature ovarian failure phenotype.
View details for DOI 10.1210/en.2003-1141
View details for Web of Science ID 000222043900048
View details for PubMedID 15059956
Bone morphogenetic proteins (BMPs) are important for body patterning and morphogenesis, whereas several BMP antagonists regulate the functions of BMPs during embryonic development and tissue differentiation. Protein related to DAN and cerberus (PRDC) is a secreted protein with a cystine knot structure identified by gene trapping in embryonic stem cells. Although PRDC shows sequence homology with proteins of the BMP antagonist family, its biological activity and physiological functions are unclear. We generated recombinant PRDC and its paralog, gremlin, and tested their ability to suppress actions initiated by diverse BMP proteins. Similar to the known BMP antagonist, gremlin, PRDC blocked ligand signaling induced by BMP2 and BMP4 but had minimal effects on reporter gene activation induced by GDF-9, activin, or transforming growth factor-beta. Co-precipitation assays further demonstrated the direct protein-protein interactions between PRDC and BMP2 or BMP4. Reverse transcriptase-PCR analyses indicated that PRDC transcripts are widely expressed showing higher levels in ovary, brain, and spleen. In mouse ovary, PRDC transcripts were increased following gonadotropin treatment. In situ hybridization analyses further indicated that ovarian PRDC transcripts are localized in granulosa cells of selective follicles. In addition, co-treatment with PRDC antagonized the inhibitory effects of BMP4 on the follicle-stimulating hormone stimulation of progesterone production by cultured rat granulosa cells. Thus, PRDC is a potent BMP antagonist with a wide tissue expression pattern, and ovarian PRDC expressed in granulosa cells could be involved in follicular development by antagonizing the actions of theca cell-derived BMPs.
View details for DOI 10.1074/jbc.M402376200
View details for Web of Science ID 000221570900043
View details for PubMedID 15039429
Mammalian oocytes are arrested at the prophase of meiosis before induction of maturation by the preovulatory luteinizing hormone (LH) surge. LH also promotes the survival of meiotic male germ cells in the testis. Because LH binds somatic cells, the mechanism underlying its regulation of germ cell function is unclear. We found that LH stimulates Leydig insulin-like 3 (INSL3) transcripts in ovarian theca and testicular Leydig cells. INSL3, in turn, binds a G protein-coupled receptor, LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8), expressed in germ cells to activate the inhibitory G protein, thus leading to decreases in cAMP production. Treatment with INSL3 initiates meiotic progression of arrested oocytes in preovulatory follicles in vitro and in vivo and suppresses male germ cell apoptosis in vivo, thus demonstrating the importance of the INSL3-LGR8 paracrine system in mediating gonadotropin actions.
View details for DOI 10.1073/pnas.0307061101
View details for Web of Science ID 000221559100023
View details for PubMedID 15123806
View details for PubMedCentralID PMC409917
Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the TGF-beta superfamily that includes TGF-beta, activin, and bone morphogenetic proteins (BMPs). GDF-9 is indispensable for the development of ovarian follicles from the primary stage, and treatment with GDF-9 enhances the progression of early follicles into small preantral follicles. Similar to other TGF-beta family ligands, GDF-9 likely initiates signaling mediated by type I and type II receptors with serine/threonine kinase activity, followed by the phosphorylation of intracellular transcription factors named Smads. We have shown previously that GDF-9 interacts with the BMP type II receptor (BMPRII) in granulosa cells, but the type I receptor involved is unknown. Using P19 cells, we now report that GDF-9 treatment stimulated the CAGA-luciferase reporter known to be responsive to TGF-beta mediated by the type I receptor, activin receptor-like kinase (ALK)5. In contrast, GDF-9 did not stimulate BMP-responsive reporters. In addition, treatment with GDF-9 induced the phosphorylation of Smad2 and Smad3 in P19 cells, and the stimulatory effect of GDF-9 on the CAGA-luciferase reporter was blocked by the inhibitory Smad7, but not Smad6. We further reconstructed the GDF-9 signaling pathway using Cos7 cells that are not responsive to GDF-9. After overexpression of ALK5, with or without exogenous Smad3, the Cos7 cells gained GDF-9 responsiveness based on the CAGA-luciferase reporter assay. The roles of ALK5 and downstream pathway genes in mediating GDF-9 actions were further tested in ovarian cells. In cultured rat granulosa cells from early antral follicles, treatment with GDF-9 stimulated the CAGA-luciferase reporter activity and induced the phosphorylation of Smad3. Furthermore, transfection with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 actions. In conclusion, although GDF-9 binds to the BMP-activated type II receptor, its downstream actions are mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins. Because ALK5 is a known receptor for TGF-beta, diverse members of the TGF-beta family of ligands appear to interact with a limited number of receptors in a combinatorial manner to activate two downstream Smad pathways.
View details for DOI 10.1210/me.2003-0393
View details for Web of Science ID 000189299100013
View details for PubMedID 14684852
View details for Web of Science ID 000220184500705
TGF-beta family proteins with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. Bone morphogenetic protein (BMP) antagonists represent a subgroup of these proteins, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these proteins were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (10-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues-gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot proteins, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.
View details for DOI 10.1210/me.2003-0227
View details for Web of Science ID 000187624600001
View details for PubMedID 14525956
View details for Web of Science ID 000222848400473
Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male. Although in Western countries 1-2% of males at the age of 3 months are diagnosed with this condition, its aetiology is still unknown. Animal models suggest a possible genetic basis for this disorder. Recently, the INSL3 (Leydig insulin-like peptide) gene and its cognate receptor, LGR8, were found to be important in testicular descent by regulating gubernacular development. Male mice null for either INSL3 or LGR8 genes exhibited bilateral cryptorchidism. Because earlier studies indicated that mutation of the INSL3 gene is not associated with the development of human cryptorchidism, this study analysed whether mutations in the LGR8 gene could be associated with this disorder. Sequencing of 18 exons of the LGR8 gene in 23 cryptorchid Finnish patients and a group of 33 control subjects allowed the identification of three nucleotide changes in exons 12 and 17, showing single base substitutions from A to G at positions 957, 993, and 1810 of LGR8. Among the three changes, only the 1810 A to G substitution is associated with an amino acid change from isoleucine to valine (Ile604Val) located in the fifth transmembrane domain of this seven-transmembrane receptor. This change was more frequent in a control group of normal fertile adult males and infant boys than in the group of cryptorchid males. The change is not associated with altered receptor signalling, thus suggesting the presence of a polymorphism unrelated to the cryptorchid phenotype. These data indicate that mutations involving the human LGR8 gene do not represent a frequent cause of cryptorchidism in the Finnish population.
View details for PubMedID 14656401
Urocortin (UCN), a member of the Corticotropin-Releasing Factor (CRF) family of peptides is a well described cardioprotective agent. UCN is able to bind to two types of G-protein coupled receptors: CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2), whereas, two homologues of UCN, stresscopin (SCP) or also known as urocortin III (UCNIII) and stresscopin related peptide (SRP), or urocortin II (UCNII), bind exclusively and with high affinity to CRFR2, we hypothesised that they will exhibit more pronounced cardioprotective effects than UCN. We show for the first time that SCP is expressed in rat cardiomyocytes and that the levels of SRP and SCP are increased by hypoxic stress. All three peptides have potent cardioprotective effects in cells exposed to hypoxia/reoxygenation. When used at 10(-8) M they increased the amount of live cells by 25% when added prior to hypoxia, and by 20% when UCN and SCP were added at the onset of reoxygenation. In addition, the peptides are equally are more potent antiapoptotic factors than UCN. The antiapoptotic effects of SCP were more pronounced than SRP and UCN at a concentration of 10(-10) M. Furthermore, SCP and SRP protect cardiomyocytes better than UCN at concentrations up to and including 10(-10) M and reduced the amount of TUNEL positive cells almost by half at concentrations of 10(-12) to 10(-10) M. More importantly, we demonstrate that SCP and SRP are able to protect cardiomyocytes even if they are administered after the hypoxic insult and prior to reoxygenation. In this case SCP was more potent than UCN and SRP at 10(-12) M and both SCP and SRP exhibited higher protection at 10(-8) M compared to UCN. Cardioprotection of cardiomyocytes by 10(-8) M of peptides was abolished when treated with 50 microM LY294002 or 100 microM PD98059, but not by 10 microM SB203580 prior to the hypoxic insult. Transfection of dominant negative Akt and MEK1 also blocked protection by the peptides, whereas dominant negative MEKK6 had no effects, demonstrating that SCP and SRP, like UCN, require activation of p42/44 Mitogen activated protein kinase and Akt/Protein Kinase B in order to produce their cardioprotective effects. In addition, we showed that SCP and UCN are potent activators of the p42/44 MAPK pathway, with SRP able to induce phosphorylation of p42/44 MAPK as well, albeit not as pronounced.
View details for DOI 10.1016/S0022-2828(03)00244-X
View details for Web of Science ID 000186037700016
View details for PubMedID 14519439
View details for Web of Science ID 000185672400074
Intercellular communication in multicellular organisms requires the relay of extracellular signals by cell surface proteins to the interiors of cells. The availability of genome sequences from humans and several model organisms has facilitated the identification of several human plasma membrane receptor families and allowed the analysis of their phylogeny. This review provides a global categorization of most known signal transduction-associated receptors as enzymes, recruiters, and latent transcription factors. The evolution of known families of human plasma membrane signaling receptors was traced in current literature and validated by sequence relatedness. This global analysis reveals themes that recur during receptor evolution and allows the formulation of hypotheses for the origins of receptors. The human receptor families involved in signaling (with the exception of channels) are presented in the Human Plasma Membrane Receptome database.
View details for PubMedID 12815191
Glycoprotein hormone receptors have ligand-binding ectodomains consisting of leucine-rich repeats, followed by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the luteinizing hormone (LH) receptor (S277I) and observed increased basal cAMP production and ligand affinity by mutant receptor. Conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of LH receptor constrains the TM region. Point mutations in the hinge region or ligand binding could cause conformational changes in the TM region that result in Gs activation.
View details for DOI 10.1016/S0303-7207(03)00075-3
View details for Web of Science ID 000183480300023
View details for PubMedID 12770743
The insulin/relaxin peptide family includes insulin, IGFs, relaxin1-3, INSL3/RLF, INSL4, INSL5/RIF2 and INSL6/RIF1, many without functional characterization. Based on analysis of transgenic phenotypes and phylogenetic profiling, we have discovered that two orphan leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8, are cognate receptors for relaxin whereas INSL3 is a specific ligand for LGR8. With the identification of the relaxin receptors, it is now possible to investigate specific cells and tissues that are responsive to relaxin in diverse physiological and pathological conditions as well as to develop agonists and antagonists for LGR7 and LGR8 as therapeutics to treat different labor disorders. Furthermore, future functional characterization of the specificity of these pluripoentent receptors with peptide ligands could lead to the understanding of related orphan ligands and receptors.
View details for DOI 10.1016/S0303-7207(03)00078-9
View details for Web of Science ID 000183480300026
View details for PubMedID 12770746
Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the transforming growth factor-beta (TGF-beta) superfamily. In GDF-9 null mice, follicle development is arrested at the primary stage and in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. In vitro, GDF-9 promotes granulosa cell proliferation but inhibits FSH-induced differentiation. GDF-9 also promotes the differentiation of theca cells in vivo and in vitro. GDF-9, like TGF-beta or activin, is a close member of the bone morphogenetic proteins (BMPs) family. GDF-9 likely initiates signaling by assembling two related but distinct types of receptors, both of which are serine/threonine kinases with a single transmembrane domain. The ligand-receptor binding activates intracellular transcription factors called Smads. In granulosa cells, Vitt et al. have shown that the BMP receptor type II is involved in GDF-9 signaling. The type I receptors and the Smad pathway for GDF-9 remain to be identified.
View details for DOI 10.1016/S0303-7207(03)00058-3
View details for Web of Science ID 000183480300007
View details for PubMedID 12770727
Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for (33)P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.
View details for DOI 10.1074/jbc.M212457200
View details for Web of Science ID 000181466800016
View details for PubMedID 12506116
Mcl-1L (myeloid cell leukemia-1 long) is an antiapoptotic Bcl-2 family protein discovered as an early induction gene during leukemia cell differentiation. Previously, we identified Mcl-1S (short) as a short splicing variant of the Mcl-1 gene with proapoptotic activity. To identify Mcl-1-interacting proteins, we performed yeast two-hybrid screening and found cDNAs encoding tankyrase 1. This protein possesses poly(ADP-ribose) polymerase activity and presumably facilitates the turnover of substrates following ADP-ribosylation. In yeast and mammalian cells, tankyrase 1 interacts with both Mcl-1L and Mcl-1S, but does not bind to other Bcl-2 family proteins tested. Analysis of truncated tankyrase 1 mutants indicated that the first 10 ankyrin repeats are involved in interaction with Mcl-1. In the N terminus of Mcl-1, a stretch of 25 amino acids is sufficient for binding to tankyrase 1. Overexpression of tankyrase 1 antagonizes both Mcl-1L-mediated cell survival and Mcl-1S-induced cell death. Furthermore, coexpression of tankyrase 1 with Mcl-1L or Mcl-1S decreased the levels of Mcl-1 proteins. Although tankyrase 1 down-regulates Mcl-1 protein expression, no ADP-ribosylation of Mcl-1 was detected. In contrast, overexpression of Mcl-1 proteins suppressed the ADP-ribosylation of the telomeric repeat binding factor 1, another tankyrase 1-interacting protein. Thus, interaction of Mcl-1L and Mcl-1S with tankyrase 1 could serve as a unique mechanism to decrease the expression of these Bcl-2 family proteins, thereby leading to the modulation of the apoptosis pathway.
View details for DOI 10.1074/jbc.M201988200
View details for Web of Science ID 000180968900108
View details for PubMedID 12475993
The TGF beta family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis. GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant GDF-9 activates components of the Smad signaling pathways known to be differentially activated by TGF beta and the bone morphogenetic proteins (BMPs). As with TGF beta, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (alpha PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral alpha PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin beta(B)-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGF beta and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary.
View details for DOI 10.1210/jc.2002-021317
View details for Web of Science ID 000180915300040
View details for PubMedID 12574210
Ovarian inhibin production is stimulated by FSH and several TGFbeta family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFbeta/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-alpha content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using (32)P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-alpha and inhibin-beta subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-alpha gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-alpha promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-alpha gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-alpha promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.
View details for DOI 10.1210/en.2002-220618
View details for Web of Science ID 000180176100023
View details for PubMedID 12488343
To determine the influence of FSH receptor variants Thr307-Asn680 (TN) and Ala307-Ser680 (AS) on ovarian function, we investigated the frequency of these gene polymorphisms by using restriction fragment length polymorphism analysis and observed their effects on clinical manifestations. In a population of 522 Japanese women, the overall frequency of TN/TN (NN), TN/AS (NS), and AS/AS (SS) was 41.0, 46.9 and 12.1% respectively. In polycystic ovary patients, the NS population was significantly larger when compared with the spontaneously ovulating group (66.7 versus 43.5%, P < 0.05). In the SS group, a significantly higher (46%) basal level of serum FSH was observed as compared with that in the NS group (P < 0.05). A higher dose of the exogenous gonadotrophin was required to achieve ovulation induction in the SS group as compared with the NS group (P < 0.05). At the time of hCG administration, estradiol levels per oocyte retrieved for IVF in the SS group were significantly lower as compared with the levels in the NS and NN groups (P < 0.05). There were no significant differences in FSH-stimulated cAMP production and PI turnover as well as ligand-binding affinity between the two receptor isoforms when overexpressed in transfected 293T cells. These results suggest that although FSH receptor polymorphisms have no discernible effect on FSH receptor function in vitro, there are associations between the genotype and some aspects of patient status.
View details for Web of Science ID 000178614500002
View details for PubMedID 12356937
Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4-8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testis Leydig cells and ovarian theca and luteal cells. Male mice mutant for INSL3 exhibit cryptorchidism or defects in testis descent due to abnormal gubernaculum development whereas overexpression of INSL3 induces ovary descent in transgenic females. Because transgenic mice missing the LGR8 gene are also cryptorchid, INSL3 was tested as the ligand for LGR8. Here, we show that treatment with INSL3 stimulated cAMP production in cells expressing recombinant LGR8 but not LGR7. In addition, interactions between INSL3 and LGR8 were demonstrated following ligand receptor cross-linking. Northern blot analysis indicated that the LGR8 transcripts are expressed in gubernaculum whereas treatment of cultured gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation. The present study identified the ligand for an orphan G protein-coupled receptor based on common phenotypes of ligand and receptor null mice. Demonstration of INSL3 as the ligand for LGR8 facilitates understanding of the mechanism of testis descent and allows studies on the role of INSL3 in gonadal and other physiological processes.
View details for DOI 10.1074/jbc.C200398200
View details for Web of Science ID 000177718700002
View details for PubMedID 12114498
Growth differentiation factor-9 (GDF-9) is a glycoprotein secreted by the oocyte that is capable of stimulating granulosa cell proliferation and inhibiting differentiation. GDF-9 is a member of the transforming growth factor beta superfamily of ligands known to signal through type I and II serine/threonine kinase receptors. In the sequenced human genome, seven type I and six type II receptors have been identified. Based on phylogenetic and sequence analyses, we predicted that GDF-9 likely interacts with known type I and type II receptors. We obtained soluble chimeric proteins with the ectodomains of candidate receptors fused to the Fc portion of immunoglobin and tested their ability to act as functional antagonists. Addition of bone morphogenetic protein receptor type II (BMPRII) ectodomain was most effective in blocking GDF-9 stimulation of granulosa cell proliferation and GDF-9 suppression of FSH-stimulated progesterone production. In addition, the ectodomains of bone morphogenetic protein receptor type IA, bone morphogenetic protein receptor type IB, and activin receptor type IIA were partially effective in blocking GDF-9 action. Furthermore, the BMPRII ectodomain directly interacted with GDF-9 in a coprecipitation study demonstrating the role of the BMPRII ectodomain as a binding protein for GDF-9. To demonstrate the role of BMPRII in GDF-9 signaling in follicular cells, the expression of this protein was blocked in cultured granulosa cells using specific BMPRII antisense oligomers. Inhibition of BMPRII biosynthesis completely prevented the GDF-9 induction of granulosa cell thymidine incorporation. GDF-9 expression is essential for early follicle development, and the presence of the type II and type I receptors in the neonatal rat ovary was verified by reverse transcription polymerase chain reaction. These results demonstrate the important role of BMPRII in mediating GDF-9 action in granulosa cells from small antral follicles and indicate that the effects of GDF-9 might be transduced by binding to BMPRII and one or more type I receptors.
View details for Web of Science ID 000177044200017
View details for PubMedID 12135884
In the postgenomic era, it is now possible to investigate the function of all human genes to provide an integrated view of physiology and pathophysiology. An organ-based approach has been used to set up a database integrating existing text-based literature on individual ovarian genes and their sequence-based data in the GenBank. The Ovarian Kaleidoscope database (OKdb) has accumulated nearly one thousand individual gene pages that are searchable based on gene function, cellular localization, chromosomal position, ovarian cell type, ovarian function, mutant phenotypes, and other criteria. The present review exemplifies the use of this organ-based database in setting up gene pathway maps for DNA array analysis, identifying key gene networks essential for infertility phenotypes, comparing chromosomal synteny regions for finding candidate fertility genes, categorizing cell-specific and hormonally coregulated genes for promoter analysis, and documenting potential ligands and receptors in the paracrine regulation of follicular development. The present global analysis of gene function and relationships in an organ-specific manner provides a functional genomic paradigm for the future understanding of the physiology and pathophysiology of diverse organs.
View details for Web of Science ID 000175653800011
View details for PubMedID 12021167
The availability of the human genomic sequence is changing the way in which biological questions are addressed. Based on the prediction of genes from nucleotide sequences, homologies among their encoded amino acids can be analyzed and used to place them in distinct families. This serves as a first step in building hypotheses for testing the structural and functional properties of previously uncharacterized paralogous genes. As genomic information from more organisms becomes available, these hypotheses can be refined through comparative genomics and phylogenetic studies. Instead of the traditional single-gene approach in endocrine research, we are beginning to gain an understanding of entire mammalian genomes, thus providing the basis to reveal subfamilies and pathways for genes involved in ligand signaling. The present review provides selective examples of postgenomic approaches in the analysis of novel genes involved in hormonal signaling and their chromosomal locations, polymorphisms, splicing variants, differential expression, and physiological function. In the postgenomic era, scientists will be able to move from a gene-by-gene approach to a reconstructionistic one by reading the encyclopedia of life from a global perspective. Eventually, a community-based approach will yield new insights into the complexity of intercellular communications, thereby offering us an understanding of hormonal physiology and pathophysiology.
View details for Web of Science ID 000176192300006
View details for PubMedID 12050125
Human thyrotropin (TSH), luteotropin (LH), follitropin (FSH), and chorionic gonadotropin are members of the heterodimeric glycoprotein hormone family. The common alpha subunit forms noncovalent heterodimers with different beta subunits. Two novel human glycoprotein hormonelike genes, alpha2 (A2) and beta5 (B5), recently have been identified. Using a yeast two-hybrid assay, the two subunits were found as potential heterodimerization partners. Immunological analyses confirmed the heterodimerization of A2 and B5 in transfected cells and their colocalization in the anterior pituitary. Recombinant A2/B5 heterodimeric glycoproteins, purified using cation exchange and size fractionation chromatography, activated human TSH receptors, but not LH and FSH receptors, and showed high affinity to TSH receptors in a radioligand receptor assay. The heterodimer also stimulated cAMP production and thymidine incorporation by cultured thyroid cells and increased serum thyroxine levels in TSH-suppressed rats in vivo. This new heterodimeric glycoprotein hormone was named as thyrostimulin based on its thyroid-stimulating activity. The expression of thyrostimulin in the anterior pituitary known to express TSH receptors suggested a paracrine mechanism. The present discovery of a new ligand based on genomic approaches could facilitate the understanding of the physiological roles of extra-thyroid TSH receptor systems and the structural-functional basis of receptor signaling by related glycoprotein hormones.
View details for DOI 10.1172/JCI200214340
View details for Web of Science ID 000176015300008
View details for PubMedID 12045258
View details for PubMedCentralID PMC150994
It is well established that LH action is mediated primarily by adenylate cyclase/cAMP. However, the role of inositol phosphate/calcium in LH signaling is less well established. We examined the effects of gonadotropins in primary culture human granulosa-lutein cells and in HEK293 cells transiently transfected with human wild-type or chimeric gonadotropin receptors. The intracellular free calcium concentration was measured using fura-2 microspectrofluorometric techniques. Human (h) LH (2-4 microg/ml) and CG (10 IU/ml) consistently evoked oscillatory calcium signals in HEK293 cells transfected with hLH receptor, whereas hFSH (2-4 microg/ml) failed to elicit any response. Conversely, both hLH and hFSH failed to elicit a calcium response from HEK293 cells transfected with hFSHR, indicating the specificity of the response to the LH receptor. Pretreatment of transfected HEK293 cells with pertussis toxin (100 ng/ml) attenuated all gonadotropin-evoked calcium mobilization. Studies with chimeric LH receptor showed that the sequence of the long extracellular portion of the receptor was not critical for stimulation of PLC activity, but maintained agonist binding specificity. The C-terminal sequence of the receptor was clearly important for the generation of the basal calcium oscillations, but the precise extent of the critical sequence has yet to be identified. Although various subdivisions of this region were capable of stimulating calcium transients, an intact carboxyl-terminal third of the receptor was required for normal and sustained intracellular calcium signaling. Our study unequivocally shows that the hLH receptor is coupled to the inositol phosphate/calcium signaling pathway via a pertussis toxin-sensitive G protein-coupled receptor.
View details for Web of Science ID 000175170500020
View details for PubMedID 11956155
Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.
View details for DOI 10.1074/jbc.M109617200
View details for Web of Science ID 000173813900023
View details for PubMedID 11723133
Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.
View details for Web of Science ID 000173560900043
View details for PubMedID 11809971
View details for Web of Science ID 000174818900015
View details for Web of Science ID 000176561900008
Growth differentiation factor-9 (GDF-9) is a growth factor and a member of the TGFbeta superfamily that is secreted by oocytes in growing ovarian follicles. In the current study we cultured human ovarian follicles within slices of ovarian cortical tissue in the presence and absence of recombinant rat GDF-9. Ovarian tissue was obtained by biopsy during cesarean section (31 women) or gynecological laparoscopy (3 women). The mean age (+/-SD) of the women who donated tissue was 32.8 +/- 5.0 yr (range, 19-41 yr). In these biopsies a total of 900 follicles were analyzed. A significantly higher proportion of cultured primordial follicles showed growth initiation and reached the secondary stage of development in the presence of GDF-9. In serum-free cultures after 7 d, 53% of the follicles had reached the secondary stage with GDF-9 vs. 31% in the control group (P < 0.01). Follicle viability was also improved in the presence of GDF-9 after 7 d in culture (74% GDF-9 vs. 48% control), which resulted in a smaller reduction in follicle numbers due to atresia. Treatment with GDF-9 in vitro promoted the survival and progression of human follicular development to the secondary stage. This oocyte-derived factor may be essential for the development of somatic cells in early human follicles and useful in designing culture conditions for maturation of follicles and oocytes in vitro.
View details for Web of Science ID 000173450700049
View details for PubMedID 11788667
View details for Web of Science ID 000174431300032
View details for Web of Science ID 000174315800001
To explore the functional role of Bcl-2 in germ cell development, transgenic mice carrying 6 kilobases of the inhibin-alpha promoter were generated to express human bcl-2 gene product in the gonads. Although female transgenic mice demonstrated decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis, the adult males exhibited variable impairment of spermatogenesis. The degree of damage ranged from tubules with intraepithelial vacuoles of varying sizes to near atrophied tubules consisting of Sertoli cells and a few spermatogonia. Although there was no significant change in body weight, an approximately 34% decrease in testicular weights was noted in transgenic animals compared with wild-type mice. Gamete maturation, assessed by determining the percentage of tubules with advanced (steps 13-16) spermatids, was decreased to 44.4% of the values measured in the wild-type animals. The incidence of germ cell apoptosis increased 3.8-fold in the transgenic animals and was associated with a marked loss of germ cells. Electron microscopy of the testes further revealed large vacuoles in the Sertoli cell cytoplasm and dilations of the intracellular spaces between adjacent Sertoli cells, spermatid malformations, and increased germ cell apoptosis in the transgenic animals. There was no evidence of Sertoli cell death either by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay or electron microscopy. Leydig cell ultrastructure, cell size and numbers, and plasma levels of testosterone were not different between normal and the transgenic animals. Collectively, these results support the critical role of Bcl-2 in male germ cell development and are consistent with the gender-specific role of the Bcl-2 family members in reproduction.
View details for Web of Science ID 000171863600010
View details for PubMedID 11700863
GDF-9 was shown to be essential for follicle progression and is the only factor secreted by the oocyte shown to increase the number of primordial and primary follicles in vivo. Furthermore, GDF-9 is a major growth factor involved in the oocyte control of granulosa cell differentiation. A concentration gradient of the paracrine factor GDF-9 established by the oocyte could provide the basis to explain the stratification of granulosa cells in antral and preovulatory follicles. The stimulatory effects of GDF-9 on early follicle development provide a basis for the use of GDF-9 in the treatment of infertility.
View details for Web of Science ID 000172077600020
View details for PubMedID 11604237
Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.
View details for Web of Science ID 000170224800001
View details for PubMedID 11483855
During the periovulatory period, the mammalian ovary is the site of dramatic functional and structural changes, leading to oocyte maturation, follicle rupture, and corpus luteum formation. To a large extent, these processes result from changes in the transcriptome of various ovarian cell types. To develop a broader view of periovulatory changes in gene expression in the ovary and to identify further genes involved in periovulatory events, we used the recently developed DNA array technology. Immature female eCG-primed rats were killed either immediately before or 6 h after ovulation induction with hCG. Total ovarian RNA was isolated and used to prepare radiolabeled cDNA probes, which were hybridized to DNA arrays representing approximately 600 rat genes. Quantitative analysis identified a multitude of regulated gene messages, including several genes involved in extracellular matrix degradation and lipid/steroid metabolism previously reported to be induced by hCG. This screening also identified a group of candidate genes whose ovarian expression and gonadotropin regulation was hitherto unknown. The induction of three of these genes, encoding cutaneous fatty acid-binding protein, the interleukin-4 receptor alpha chain, and prepronociceptin, was confirmed and further characterized by Northern blot analysis. In addition, in situ hybridization analysis showed that hCG administration resulted in exclusive or predominant expression of all three genes in theca cells. These results demonstrate that DNA arrays can be used to identify genes regulated during the periovulatory period, thus contributing to a more detailed understanding of the molecular mechanisms of ovulation.
View details for Web of Science ID 000169515800034
View details for PubMedID 11420249
P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6-9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin-1beta, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process.
View details for Web of Science ID 000168903100022
View details for PubMedID 11356677
Adaptive stress responses mediated by the endocrine, autonomic, cardiovascular and immune systems are essential for the survival of the individual. Initial stress-induced responses provide a vital short-term metabolic lift, but prolonged or inappropriate exposure to stress can compromise homeostasis thereby leading to disease. This 'fight-or-flight' response is characterized by the activation of the corticotropin-releasing hormone (CRH)-adrenocorticotropin-glucocorticoid axis, mediated by the type 1 CRH receptor. In contrast, the type 2 CRH receptor mediates the stress-coping responses during the recovery phase of stress. We identified human stresscopin (SCP) and stresscopin-related peptide (SRP) as specific ligands for the type 2 CRH receptor. The genes encoding these peptides were expressed in diverse peripheral tissues as well as in the central nervous system. Treatment with SCP or SRP suppressed food intake, delayed gastric emptying and decreased heat-induced edema. Thus SCP and SRP might represent endogenous ligands for maintaining homeostasis after stress, and could allow the design of drugs to ameliorate stress-related diseases.
View details for Web of Science ID 000169961100041
View details for PubMedID 11329063
The cystine knot three-dimensional structure is found in many extracellular molecules and is conserved among divergent species. The identification of proteins with a cystine knot structure is difficult by commonly used pairwise alignments because the sequence homology among these proteins is low. Taking advantage of complete genome sequences in diverse organisms, we used a complementary approach of pattern searches and pairwise alignments to screen the predicted protein sequences of five model species (human, fly, worm, slime mold, and yeast) and retrieved proteins with low sequence homology but containing a typical cystine knot signature. Sequence comparison between proteins known to have a cystine knot three-dimensional structure (transforming growth factor-beta, glycoprotein hormone, and platelet-derived growth factor subfamily members) identified new crucial amino acid residues (two hydrophilic amino acid residues flanking cysteine 5 of the cystine knot). In addition to the well known members of the cystine knot superfamily, novel subfamilies of proteins (mucins, norrie disease protein, von Willebrand factor, bone morphogenetic protein antagonists, and slit-like proteins) were identified as putative cystine knot-containing proteins. Phylogenetic analysis revealed the ancient evolution of these proteins and the relationship between hormones [e.g. transforming growth factor-beta (TGFbeta)] and extracellular matrix proteins (e.g. mucins). They are absent in the unicellular yeast genome but present in nematode, fly, and higher species, indicating that the cystine knot structure evolved in extracellular signaling molecules of multicellular organisms. All data retrieved by this study can be viewed at http://hormone.stanford.edu/.
View details for Web of Science ID 000168393200001
View details for PubMedID 11328851
Concentrations of follicle-stimulating hormone (FSH) have an important role in multiple ovulation. An association has been reported between mutations in the FSH receptor (FSHR) in a family with increased twinning frequency. We sequenced the transmembrane region of FSHR (located on chromosome 2) in 21 unrelated mothers of dizygotic twins and found no differences to the published sequence. A linkage study of 183 sister pairs and trios, in which all sisters had given birth to spontaneous dizygotic twins, excluded linkage to this region of chromosome 2. We conclude that mutations in FSHR are not a common cause of familial dizygotic twinning.
View details for Web of Science ID 000167400900020
View details for PubMedID 11253976
Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.
View details for Web of Science ID 000166200200035
View details for PubMedID 11133686
View details for Web of Science ID 000172402100030
The entire human genome will be sequenced in September 2000. Facing the exponential increase of data in the GenBank at the National Center for Biotechnology Information, reproductive biologists are being bombarded with massive amounts of information on diverse genes. It is becoming increasingly difficult for individual investigators to sort out the diverse genetic and physiologic information on the localization and function of different genes in the ovary. To alleviate the present situation, we have taken advantage of the accessibility of the Internet and initiated a project that serves the entire ovarian research community.The Ovarian Kaleidoscope database provides information regarding biologic function, expression pattern, and regulation of genes that are expressed in the ovary. In addition, it serves as a gateway to other online information resources relevant to ovarian research by offering results from original papers and data about nucleotide and amino acid sequences, and human and murine mutation phenotypes. All references are linked by hypertext to PubMed and additional links to sequence databases are also included. This information is accessible online and searchable not only by gene name but also by criteria such as the cellular and ovarian function of the gene product, the expression of genes in different ovarian cell types, or their association with specific ovarian phenotypes.
View details for Web of Science ID 000167356900012
View details for PubMedID 11223370
The receptors for lutropin (LH), FSH, and TSH belong to the large G protein-coupled receptor (GPCR) superfamily and are unique in having a large N-terminal extracellular (ecto-) domain important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals. Based on the sequences of mammalian glycoprotein hormone receptors, we have identified a new LGR in Drosophila melanogaster and named it as fly LGR2 to distinguish it from the previously reported fly LH/FSH/TSH receptor (renamed as fly LGR1). Genomic analysis indicated the presence of 10 exons in fly LGR2 as compared with 16 exons in fly LGR1. The deduced fly LGR2 complementary DNA (cDNA) showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Comparison of 12 LGRs from diverse species indicated that these proteins can be divided into three subfamilies and fly LGR1 and LGR2 belong to different subfamilies. Potential signaling mechanisms were tested in human 293T cells overexpressing the fly receptors. Of interest, fly LGR1, but not LGR2, showed constitutive activity as reflected by elevated basal cAMP production in transfected cells. The basal activity of fly LGR1 was further augmented following point mutations of key residues in the intracellular loop 3 or transmembrane VI, similar to those found in patients with familial male precocious puberty. The present study reports the cloning of fly LGR2 and indicates that the G protein-coupling mechanism is conserved in fly LGR1 as compared with the mammalian glycoprotein hormone receptors. The characterization of fly receptors with features similar to mammalian glycoprotein hormone receptors allows a better understanding of the evolution of this unique group of GPCRs and future elucidation of their ligand signaling mechanisms.
View details for Web of Science ID 000089970300020
View details for PubMedID 11089539
Growth differentiation factor (GDF)-9 is a cystine knot-containing hormone of the transforming growth factor-beta superfamily produced by the oocyte. In GDF-9 null mice, follicle development is arrested at the primary stage and GDF-9 treatment in vitro enhances preantral follicle growth. Immature female rats were treated with recombinant GDF-9 for 7 or 10 days. At 10 days, treatment with GDF-9 augmented ovarian weights, concomitant with an increase in the number of primary and small preantral follicles by 30 and 60%, respectively. Furthermore, the number of primordial follicles was decreased by 29%, but the number of large preantral follicles was not affected. In contrast, treatment with FSH increased the number of small and large preantral follicles by 36 and 177% but did not influence the number of primary and primordial follicles. Immunoblot analysis showed an increase of CYP17, a theca cell marker, in the ovarian homogenate after treatment with GDF-9 but not FSH. The present results indicate that in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. Thus, GDF-9 treatment could provide an alternative approach to stimulate early follicle development in addition to the widely used FSH that acts mainly on the development of more advanced follicles.
View details for Web of Science ID 000089394800035
View details for PubMedID 11014238
Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.
View details for Web of Science ID 000089489300034
View details for PubMedID 10993847
Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.
View details for Web of Science ID 000089577900049
View details for PubMedID 10889210
The Ovarian Kaleidoscope database (OKdb) is a collaborative online resource for scientists investigating the ovary. It provides information regarding the biological function, expression pattern, and regulation of genes expressed in the ovary as well as for the phenotypes associated with their mutation. In addition, the records in the OKdb are linked to other sites offering online information about biomedical publications, nucleotide and amino acid sequences, and human genes and genetic disorders. A powerful search tool allows the retrieval of records for specific genes and gene products based on their properties at the molecular, cellular, ovarian, or organism level. Researchers working on particular aspects of ovarian physiology can submit information into the database through a simple web-based form and instantly update their records as additional data become available. Because of this approach, the OKdb website could serve as a tool with which to navigate through the rapidly expanding amount of information about the expression and function of individual genes in the ovary and could also enhance communication within the ovarian research community. Moreover, the design of the OKdb could serve as a model for the development of other online databases of tissue-specific gene expression and function. The OKdb can be accessed at http://ovary.stanford.edu/.
View details for Web of Science ID 000088872200002
View details for PubMedID 10965872
Natural multiple pregnancy in women leading to dizygotic (DZ) twins is familial and varies across racial groups, suggesting a genetic predisposition. Mothers of DZ twins have a higher incidence of spontaneous multiple ovulation and elevated FSH concentrations. FSH release is controlled by feedback of inhibin peptides from the ovary, and immunization against inhibin alpha-subunit results in an increased ovulation rate in animals. The inhibin alpha-subunit is therefore a candidate gene for mutations that may increase the frequency of DZ twinning. Restriction digests of a PCR product from exon 1 with the enzyme SpeI detects a C/T polymorphism at bp 128 with two alleles of 447 and 323/124 bp. The polymorphism was typed in 1,125 individuals from 326 pedigrees with 717 mothers of spontaneous DZ twins. The alpha-inhibin locus mapped within 3 centimorgans of D2S164, and linkage with DZ twinning was excluded [decimal log odds ratio (LOD) score, -2.81 at theta = 0]. There was complete exclusion of linkage (LOD, less than -2) of a gene conferring relative risk 1.8 (lambdas, >1.8) across the chromosome, except at the p-terminus region and a small peak (maximum LOD score, 0.6) in the region of D2S151-D2S326. Analysis using either recessive or dominant models excluded linkage with DZ twinning in this population (LOD score, less than -2.5) across chromosome 2. We conclude that dizygotic twinning is not linked to variation in the alpha-inhibin locus. The results also suggest that mutations in other candidates on chromosome 2, including the receptor for FSH and the betaB-inhibin subunit (INHBB) cannot be major contributors to risk for DZ twinning.
View details for Web of Science ID 000089166200065
View details for PubMedID 10999839
MCL-1 (myeloid cell leukemia-1) is an antiapoptotic BCL-2 family protein discovered as an early induction gene during myeloblastic leukemia cell differentiation. This survival protein has the BCL-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region. We identified a short splicing variant of the MCL-1 mRNA in the human placenta encoding a protein, termed MCL-1 short (MCL-1S), with an altered C terminus as compared with the full-length MCL-1 long (MCL-1L), leading to the loss of BH1, BH2, and the transmembrane domains. Analysis of the human MCL-1 gene indicated that MCL-1S results from the splicing out of exon 2 during mRNA processing. MCL-1S, unlike MCL-1L, does not interact with diverse proapoptotic BCL-2-related proteins in the yeast two-hybrid system. In contrast, MCL-1S dimerizes with MCL-1L in the yeast assay and coprecipitates with MCL-1L in transfected mammalian cells. Overexpression of MCL-1S induces apoptosis in transfected Chinese hamster ovary cells, and the MCL-1S action was antagonized by the antiapoptotic MCL-1L. Thus, the naturally occurring MCL-1S variant represents a new proapoptotic BH3 domain-only protein capable of dimerizing with the antiapoptotic MCL-1L. The fate of MCL-1-expressing cells could be regulated through alternative splicing mechanisms and interactions of the resulting anti- and proapoptotic gene products.
View details for Web of Science ID 000088849400030
View details for PubMedID 10837489
Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.
View details for Web of Science ID 000088498400012
View details for PubMedID 10935549
Although earlier studies focused on the hormonal regulation of antral and preovulatory follicles, recent studies indicate the importance of the hormonal control mechanism for preantral follicles. The endocrine hormone FSH is not only a survival factor for early antral follicles but also a potent growth and differentiation factor for preantral follicles. In addition, KGF secreted by theca cells and c-kit ligand secreted by granulosa cells play paracrine roles in the regulation of preantral follicle growth and development. Furthermore oocyte-derived GDF-9 promotes the growth and differentiation of early follicles by acting on somatic cells in the follicle. It is likely that the genetic makeup of an oocyte could determine the secretion of oocyte hormones which would, in turn, regulate the growth and differentiation of the surrounding somatic cells of that follicle. A better understanding of the hormonal mechanisms underlying early follicle development could provide a refined culture system for the in vitro maturation of fertilizable oocytes and future design of fertility and contraceptive agents.
View details for Web of Science ID 000088889300015
View details for PubMedID 10963880
Apoptosis is an essential physiological process by which multicellular organisms eliminate superfluous cells. An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals. The Bcl-2 proteins can be divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain. In the mammalian ovary, a high rate of follicular cell apoptosis continues during reproductive life. With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes serves as a paradigm to understand apoptosis regulation in a tissue-specific manner. We identified Mcl-1 as the main ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands. The activity of the proapoptotic ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14-3-3 or hormone-induced P11. In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and, together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs and caspases. Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic approaches for pathological states.
View details for Web of Science ID 000086032300002
View details for PubMedID 10747202
Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. The dynamics of ovarian follicle development have interested endocrinologists and developmental biologists for many years. With the advent of assisted reproductive techniques in humans, the possibility of regulating follicle development in vivo and in vitro has gained clinical relevance. In this review, we focus upon key branching points during the development of ovarian follicles as well as factors involved in determining the eventual destiny of individual follicles. We discuss inconsistencies in the literature regarding the definitions of follicle recruitment and selection and propose to name the two major steps of follicle development as initial and cyclic recruitment, respectively. Because some of these disparities have arisen due to differences in the animal systems studied, we also compare the development of the ovarian follicles of both humans and rats. We also review the status of knowledge of several puzzling clinical issues that may provide important clues toward unlocking the mechanisms of follicle development.
View details for Web of Science ID 000086493300004
View details for PubMedID 10782364
In addition to pituitary gonadotropins and paracrine factors, ovarian follicle development is also modulated by oocyte factors capable of stimulating granulosa cell proliferation but suppressing their differentiation. The nature of these oocyte factors is unclear. Because growth differentiation factor-9 (GDF-9) enhanced preantral follicle growth and was detected in the oocytes of early antral and preovulatory follicles, we hypothesized that this oocyte hormone could regulate the proliferation and differentiation of granulosa cells from these advanced follicles. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells from both early antral and preovulatory follicles, accompanied by increases in granulosa cell number. Although GDF-9 treatment alone stimulated basal steroidogenesis in granulosa cells, cotreatment with GDF-9 suppressed FSH-stimulated progesterone and estradiol production. In addition, GDF-9 cotreatment attentuated FSH-induced LH receptor formation. The inhibitory effects of GDF-9 on FSH-induced granulosa cell differentiation were accompanied by decreases in the FSH-induced cAMP production. These data suggested that GDF-9 is a proliferation factor for granulosa cells from early antral and preovulatory follicles but suppresses FSH-induced differentiation of the same cells. Thus, oocyte-derived GDF-9 could account, at least partially, for the oocyte factor(s) previously reported to control cumulus and granulosa cell differentiation.
View details for Web of Science ID 000084987400020
View details for PubMedID 10642575
The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.
View details for Web of Science ID 000085163500006
View details for PubMedID 10674399
View details for Web of Science ID 000165406800015
View details for Web of Science ID 000086346600078
The majority of ovarian follicles undergo atresia mediated by apoptosis. Bcl-2-related proteins act as regulators of apoptosis via the formation of dimers with proteins inside and outside the Bcl-2 family. Previous studies have identified BAD as a proapoptotic Bcl-2 family member expressed in the ovary. It is known that BAD phosphorylation induced by survival factors leads to its preferential binding to 14-3-3 and suppression of the death-inducing function of BAD. To identify ovarian binding partners for hypophosphorylated BAD, we performed a yeast two-hybrid screening of a rat ovary complementary DNA library using as bait a mutant BAD incapable of binding to 14-3-3. Screening of yeast transformants yielded positive clones encoding the rat ortholog of Mcl-1 (myeloid cell leukemia-1), an antiapoptotic Bcl-2 protein. Amino acid sequence analysis revealed that rat and human Mcl-1 showed a complete conservation of the Bcl-2 homology domains BH1, BH2, and BH3. In the yeast two-hybrid system, Mcl-1 binds to the hypophosphorylated mutant of BAD and interacts preferentially with different proapoptotic (Bax, Bak, Bok, Bik, and BOD) compared with antiapoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, CED-9, and BHRF-1). Northern blot hybridization demonstrated expression of Mcl-1 transcripts of 2.3 and 3.7 kb in the ovary and diverse other rat tissues. In immature rats, PMSG treatment led to a transient increase in the 2.3-kb Mcl-1 transcript, peaking at 6 h after injection and returning to baseline levels after 24 h. Moreover, the same transcript was induced in the PMSG-primed preovulatory rat ovary 6 h after the administration of ovulatory doses of either hCG or FSH. In situ hybridization studies revealed that the gonadotropin stimulation of ovarian Mcl-1 message occurs in both granulosa and thecal cells. In conclusion, rat Mcl-1 was identified as an ovarian BAD-interacting protein and the message for the antiapoptotic Mcl-1 protein was induced after treatment with gonadotropins in granulosa and thecal cells of growing follicles.
View details for Web of Science ID 000083792500002
View details for PubMedID 10579309
Human CG (hCG) is a member of the glycoprotein hormone family characterized by a heterodimeric structure consisting of a common alpha-subunit noncovalently bound to a hormone-specific beta-subunit. The two subunits are highly intertwined and only the heterodimer is functional, implying that the quaternary structure is critical for biological activity. To assess the dependence of the bioactivity of hCG on the heterodimeric interactions, alpha- and beta-subunits bearing mutations that prevent assembly were covalently linked to form a single chain hCG. Receptor binding and signal transduction of these analogs were tested and their structural integrity analyzed using a panel of monoclonal antibodies (mAbs). These included dimer-specific mAbs, which react with at least four different epitope sites on the hormone, and some that react only with the free beta-subunit. We showed that there was significant loss of quaternary and tertiary structure in several regions of the molecule. This was most pronounced in single chains that had one of the disulfide bonds of the cystine knot disrupted in either the alpha- or beta-subunit. Despite these structural changes, the in vitro receptor binding and signal transduction of the single chain analogs were comparable to those of the nonmutated single chain, demonstrating that not all of the quaternary configuration of the hormone is necessary for biological activity.
View details for Web of Science ID 000084093600016
View details for PubMedID 10598590
To determine the effect of treatment with keratinocyte growth factor (KGF) on the survival of cells in cultured preantral follicles and on the growth and differentiation of preantral follicles.Preantral follicles (140-150 microm) were dissected mechanically from the ovaries of 14-day-old rats and cultured for 24 hours with and without KGF. Genomic DNA was extracted, labeled with [32P]-dideoxyadenosine triphosphate, and fractionated through agarose gels. For growth studies, the follicles were cultured individually in 96-well dishes. After 72 hours, the follicles were collected and their protein or DNA content was evaluated and their inhibin-alpha content was determined.Keratinocyte growth factor suppressed apoptosis in cultured preantral follicles by 60%. Treatment with KGF or FSH increased follicle diameter by 8% and 16%, respectively, and combined treatment with KGF and FSH increased follicle diameter by 26%. Western blot analysis demonstrated increased expression of inhibin-alpha content after treatment with KGF (2-fold), treatment with FSH (4-fold), and combined treatment with FSH and KGF (12-fold), demonstrating the effect of KGF on preantral follicle differentiation.Treatment with KGF promotes the survival, growth, and differentiation of cultured preantral follicles. Keratinocyte growth factor produced by theca cells may play a role in the progression of early follicle development.
View details for Web of Science ID 000079355700025
View details for PubMedID 10202888
Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9) gene are characterized by the arrest of ovarian follicle development at the primary stage. Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and generated recombinant GDF-9 protein to study its physiological role. Using bacteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, specific antibodies to the mature form of GDF-9 was generated. Immunohistochemical staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immunoblotting demonstrated the secretion of GDF-9 by mammalian cells transfected with GDF-9 cDNAs. Recombinant GDF-9 was shown to be an N-glycosylated protein capable of stimulating early follicle development. Growth of preantral follicles isolated from immature rats was enhanced by treatment with either GDF-9 or FSH whereas the combined treatment showed an additive effect. In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-alpha content in explants of neonatal ovaries. In contrast, the stimulatory effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was apparently not bioactive. Thus, the present study demonstrates the important role of GDF-9 in early follicle growth and differentiation. The availability of recombinant bioactive GDF-9 allows future studies on the physiological role of GDF-9 in ovarian development in vivo.
View details for Web of Science ID 000078733700029
View details for PubMedID 10067849
WT1 is a zinc finger protein with transcriptional repressor activity on several growth factor and growth factor receptor genes. In the ovary, a potential role for WT1 in the suppression of the development of immature follicles has been demonstrated. Here, gel retardation assays further showed that recombinant WT1 protein interacted with consensus DNA sequences in the inhibin-alpha gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1-5 mm in diameter) and its absence in small yellow (6-12 mm in diameter) or larger follicles (F1-F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.
View details for Web of Science ID 000078380900024
View details for PubMedID 9916003
The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interaction with the glycoprotein ligands. We have identified two new leucine-rich repeat-containing, G protein-coupled receptors and named them as LGR4 and LGR5, respectively. The ectodomains of both receptors contain 17 leucine-rich repeats together with N- and C-terminal flanking cysteine-rich sequences, compared with 9 repeats found in known glycoprotein hormone receptors. The leucine-rich repeats in LGR4 and LGR5 are arrays of 24 amino acids showing similarity to repeats found in the acid labile subunit of the insulin-like growth factor (IGF)/IGF binding protein complexes as well as slit, decorin, and Toll proteins. The TM region and the junction between ectodomain and TM 1 are highly conserved in LGR4, LGR5, and seven other LGRs from sea anemone, fly, nematode, mollusk, and mammal, suggesting their common evolutionary origin. In contrast to the restricted tissue expression of gonadotropin and TSH receptors in gonads and thyroid, respectively, LGR4 is expressed in diverse tissues including ovary, testis, adrenal, placenta, thymus, spinal cord, and thyroid, whereas LGR5 is found in muscle, placenta, spinal cord, and brain. Hybridization analysis of genomic DNA indicated that LGR4 and LGR5 genes are conserved in mammals. Comparison of overall amino acid sequences indicated that LGR4 and LGR5 are closely related to each other but diverge, during evolution, from the homologous receptor found in snail and the mammalian glycoprotein hormone receptors. The identification and characterization of new members of the LGR subfamily of receptor genes not only allow future isolation of their ligands and understanding of their physiological roles but also reveal the evolutionary relationship of G protein-coupled receptors with leucine-rich repeats.
View details for Web of Science ID 000077212900003
View details for PubMedID 9849958
Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further indicated the expression of DEFT mRNA in meiotic male germ cells. In a model of germ cell apoptosis induction, an increase in testis DEFT mRNA was found in immature rats after 2 days of treatment with a GnRH antagonist. Unlike FADD and procaspase-8/FLICE, overexpression of DEFT did not induce apoptosis in Chinese hamster ovary cells. Although cotransfection studies indicated that DEFT is incapable of modulating apoptosis effected by FADD and procaspase-8/FLICE, interactions between DEFT and uncharacterized DED-containing molecules in the testis remain to be studied in the future. In conclusion, we have identified a novel DED-containing protein with high expression in testis germ cells. This protein may be important in the regulation of death factor-induced apoptosis in the testis and other tissues.
View details for Web of Science ID 000077104000011
View details for PubMedID 9832420
Bok (Bcl-2-related ovarian killer) is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. In addition to the Bcl-2 homology (BH) domains 1 and 2 and the transmembrane sequence, Bok also has a BH3 domain believed to be important for dimerization with selective antiapoptotic Bcl-2 proteins and cell killing. We identified a splicing variant of Bok mRNA with a deletion of 43 residues from the full-length protein (Bok-L), leading to the fusion of the N-terminal-half of its BH3 domain to the C-terminal-half of the BH1 domain. Genomic analysis indicated that the Bok has five exons, and the short form of Bok (Bok-S) represents the splicing out of exon three during transcription. Although Bok-S retains the apoptosis-inducing activity in transfected cells, it has lost the ability to dimerize with antiapoptotic proteins in vitro. Additional BH3 domain mutations of Bok-L also led to defective heterodimerization without affecting its proapoptotic action. Furthermore, similar deletions for the related channel-forming proapoptotic Bax and Bak did not impair their cell killing ability. Thus, the naturally occurring Bok-S variant represents a new form of proapoptotic protein that induces cell killing without heterodimerization with antiapoptotic Bcl-2 proteins. This variant appears to contain the minimal module spanning BH1 and BH2 domains and the transmembrane sequence for apoptosis induction by channel-forming Bcl-2 proteins.
View details for Web of Science ID 000077008100016
View details for PubMedID 9804769
View details for Web of Science ID 000076906701247
Apoptosis is an active cell 'suicide' essential for the elimination of superfluous cells during diverse physiological processes in essentially all animal species. Although regulation of apoptosis by extracellular mediators is cell type-specific, new insights based on characterization of conserved intracellular effectors have suggested that intracellular pathways leading to apoptosis in diverse organisms is regulated by a group of evolutionarily conserved genes including ced-9/Bcl-2, ced-4/Apaf-1 and ced3/caspases gene families. To study whether the Bcl-2 family proteins are important in the regulation of ovarian cell apoptosis, we have used transgenic mice and yeast 2-hybrid protein protein interaction assay to characterize the roles of Bcl-2 family proteins in ovarian atresia. The use of 2-hybrid analysis resulted in the isolation of a novel pro-apoptotic Bcl-2 protein, Bcl-2-related ovarian killer (Bok) and the identification of upstream mediators for ovarian cell apoptosis.
View details for Web of Science ID 000077723300005
View details for PubMedID 9922095
Using the yeast two-hybrid protein-protein interaction system to search for genes capable of forming dimers with the antiapoptotic protein Mcl-1, we have isolated BOD (Bcl-2-related ovarian death agonist) from an ovarian fusion cDNA library. The three variants of BOD (long, medium, and short) have an open reading frame of 196, 110, and 93 amino acids, respectively; all of them contain a consensus Bcl-2 homology 3 (BH3) domain but lack other BH domains found in channel-forming Bcl-2 family proteins. In the yeast cell assay, BOD interacts with diverse antiapoptotic Bcl-2 proteins [Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1, and Epstein-Barr virus (EBV) BHRF-1] but not with different proapoptotic Bcl-2 proteins (BAD, Bak, Bok, and Bax). After overexpression in mammalian Chinese hamster ovary (CHO) cells, BOD induces apoptosis that can be prevented by the baculoviral caspase inhibitor P35. The cell-killing activity of BOD is also antagonized in cells cotransfected with the antiapoptotic Bcl-w protein, which showed high affinity for BOD in the two-hybrid assay. Furthermore, mutagenesis studies showed that BOD mutants with alterations in the BH3 domain lose cell-killing ability, suggesting that the BH3 domain is important for the mediation of cell killing by BOD. BOD mRNA is ubiquitously expressed in ovary and multiple other tissues. The BOD gene is also conserved in diverse mammalian species. Identification of BOD expands the group of proapoptotic Bcl-2 proteins that only contains the BH3 domain and allows future elucidation of the intracellular mechanism for apoptosis regulation in ovary and other tissues.
View details for Web of Science ID 000075601200014
View details for PubMedID 9731710
It has become evident that the physiological removal of cells through apoptosis during embryonic and postnatal development of multicellular organisms is a mandatory process to maintain a homeostatic state of the individual. In the ovary, massive cell death occurs during neonatal and postnatal life as an integral part of the normal ovarian development. At birth, mammalian ovaries are endowed with a fixed number of non-growing follicles that will be gradually recruited into a growing pool during reproductive life. Once follicles start growth they are either selected for ovulation or, for the majority of them, removed by apoptosis. Thus, removal of excess ovarian cells by apoptosis is necessary for normal development of the ovary. Despite the important role of follicle atresia in the maintenance of normal follicle development, studies on the hormonal control of follicle cell demise during follicle growth have not been possible until the recent development of apoptosis detection methods. Recent biochemical analysis has revealed the occurrence of internucleosomal DNA fragmentation, a hallmark of apoptosis, in atretic follicles and has facilitated the investigation into the intra-ovarian hormonal regulation of follicle atresia. This review summarizes the recent advances in the intra-ovarian hormonal mechanisms that control follicle apoptosis.
View details for Web of Science ID 000075985300005
View details for PubMedID 9786453
During each reproductive cycle, a preovulatory surge of gonadotropins induces meiotic maturation of the oocyte in the preovulatory follicle followed by ovulation. Although gonadotropins stimulate cAMP production in somatic cells of the follicle, a decrease in intra-oocyte cAMP levels is required for resumption of meiosis in oocytes. Based on the observed compartmentalization of the cAMP-degrading enzyme, phosphodiesterase, in follicular somatic and germ cells, inhibitors of phosphodiesterase 3 were used to block meiosis in ovulating oocytes in rodents. By this strategy, we demonstrated that fertilization and pregnancy could be prevented without disturbing follicle rupture and normal estrous cyclicity. In contrast to conventional contraceptive pills that disrupt ovarian steroidogenesis and reproductive cycles, the present strategy achieves effective contraception by selective blockage of oocyte maturation and development without alterations in ovulation and reproductive cyclicity.
View details for Web of Science ID 000075327900009
View details for PubMedID 9691090
View details for PubMedCentralID PMC508914
Mammalian germ cells arise in the yolk sac endoderm at the caudal aspect of the embryo and migrate to the mesodermally-derived gonadal ridge early in development. After the oogonia reach the gonadal ridge, the process of meiosis begins which coincides with the first major wave of apoptosis of female germ cells (Coucouvanis et al., 1993). Subsequently, oocytes progress to the dictyate stage of prophase I where they remain arrested until ovulation.
View details for Web of Science ID 000075156000004
View details for PubMedID 9722162
TSH and the gonadotropins, FSH, LH, and CG are a family of heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a hormone specific beta-subunit. Assembly of alpha- and beta-subunits is essential for hormone-specific posttranslational modifications, receptor binding, and bioactivity. Structure-function studies of TSH and gonadotropins using site-directed mutagenesis can often affect folding, assembly, and secretion of the hormone. To circumvent these difficulties, recently, the gonadotropin heterodimers were converted to single chains. Here we converted the hTSH heterodimer to a biologically active single chain by genetically fusing the amino terminal end of the common alpha-subunit to the carboxyl terminal end of hTSHbeta in the presence or absence of hCGbeta carboxyl terminal peptide (CTP), which was used as a linker. Wild-type hTSH and the single chains were expressed in Chinese hamster ovary (CHO) cells, and they were efficiently secreted. Although the secretion rate of the single chain was 3-fold higher than that of hTSH wild-type. Moreover, the secretion of the single chain in the presence of the CTP linker was dramatically increased. On the other hand, receptor binding and in vitro bioactivity of the single chains were similar to that of hTSH wild-type. These data indicate the potential of the single chain approach to further investigate structure-function relationships of TSH.
View details for Web of Science ID 000073367100037
View details for PubMedID 9564858
A soluble form of the amino-terminal extracellular (ecto-) domain of the human TSH receptor was generated. This protein was capable of binding TSH and autoimmune antibodies found in Graves' patients. A deletion mutant of the ectodomain lacking nine amino acids in the C-terminal region lost its ability to interact with TSH but retained binding to Graves' IgGs. In cells expressing recombinant TSH receptors, cotreatment with the mutant protein blocked the cAMP production induced by stimulating antibodies from all Graves' patients tested but was without effect on TSH action. The ability to dissociate the actions of TSH and Graves' IgGs provides a tool with which to study the mechanisms underlying Graves' disease and the possibility of neutralizing the undesirable effects of thyroid-stimulating antibodies without altering the normal responses to TSH.
View details for Web of Science ID 000071491000034
View details for PubMedID 9449640
View details for Web of Science ID 000074546000056
Bcl-2-related anti- and proapoptotic proteins are important in the decision step of the intracellular death program upstream from the caspase proteases. Targeted overexpression of Bcl-2 in ovarian somatic cells of transgenic mice leads to decreased apoptosis of granulosa cells and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation. The ability of exogenous Bcl-2 proteins to promote follicle cell survival suggests that the transgene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellular apoptosis process in favor of cell survival. We used the yeast two-hybrid system to search for ovarian Bcl-2 interacting proteins. The screening of an ovarian fusion complementary DNA library yielded several clones encoding for the death agonist Bcl-XL/Bcl-2-associated death promoter (BAD). Dimerization of Bcl-2-related proteins mediated by the consensus Bcl-2 homology (BH) domains is essential for their apoptosis-regulating function. Consistent with these observations, yeast two-hybrid assays indicated that the interaction of Bcl-2 with BAD is dependent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis showed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hybridization analysis indicated BAD mRNA expression in granulosa cells of different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 days and did not alter during the developmentally occurring apoptosis found about postnatal day 18 when the first group of early antral follicles were formed. Similarly, BAD mRNA levels did not change during follicle atresia induced by estrogen withdrawal in immature rats. To study the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell line. Overexpression of BAD induced apoptosis in both cell types, and the effect of BAD was reversed by a membrane-permeable caspase inhibitor, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Bcl-2-interacting protein in the ovary, and BAD mRNA is constitutively expressed in granulosa cells, suggesting that BAD is an essential part of the ovarian cell death process. Because BAD overexpression in granulosa cells leads to apoptosis, future studies on ovarian BAD binding proteins and hormonal regulation of the interactions among different Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.
View details for Web of Science ID A1997YH19000053
View details for PubMedID 9389536
In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.
View details for Web of Science ID A1997YF39300030
View details for PubMedID 9356461
View details for PubMedCentralID PMC24966
Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.
View details for Web of Science ID A1997YD56300011
View details for PubMedID 9369453
The stimulatory effects of gonadotropins on antral and preovulatory follicles are well known, but conflicting results have been reported regarding the gonadotropin responsiveness and dependency of preantral follicles. Taking advantage of the relatively uniform development of the first wave of follicles in the postnatal rat ovary, we evaluated the role of endogenous and exogenous gonadotropins on preantral follicle development. Reduction of the high levels of gonadotropins present in juvenile rats by either hypophysectomy (at Day 15) or GnRH antagonist treatment (starting from Day 11 of age) resulted in decreased ovarian weight at Day 19 of age that was associated with a reduced number of developing follicles and increased atresia of remaining follicles. In contrast, treatment with FSHctp (a long-acting FSH agonist) in intact (Days 5-19 of age), hypophysectomized (Days 15-19), or GnRH antagonist-treated (Days 11-19) animals resulted in increased ovarian weight and follicle development as determined histologically and by inhibin-alpha expression. A dose-dependent stimulatory effect of hCG on ovarian weight was seen when animals were cotreated with FSHctp and the GnRH antagonist. At low doses of hCG, augmentation of antral follicle formation occurred, whereas higher doses of hCG led to morphological signs of luteinization. These findings demonstrate the important role of endogenous gonadotropins in preantral follicle development and indicate that preantral follicles are highly responsive to exogenous gonadotropins.
View details for Web of Science ID A1997YD11400006
View details for PubMedID 9369162
Gonadotropin receptors are unique members of the seven-transmembrane (TM), G protein-coupled receptor family with a large extracellular (EC) sequence forming the high-affinity ligand binding domain. In a patient with Leydig cell hypoplasia, we identified a mutant LH receptor that is truncated at TM5. This protein retains limited ligand binding ability but cannot mediate cAMP responses. To study interactions between receptor fragments defective in either ligand binding or signal transduction, we co-expressed this truncated receptor together with a chimeric receptor containing the EC region of the FSH receptor and the TM region of the LH receptor. Although the chimeric receptor could not respond to human chorionic gonadotropin in producing cAMP, co-expression with the truncated LH receptor allowed partial restoration of ligand signaling through intermolecular interactions. In addition, co-expression of the same truncated LH receptor with an N-terminally truncated LH receptor that lacked the EC ligand binding domain also partially restored ligand signaling. Further shortening of the TM region in the mutant receptor found in the patient indicated that the EC domain and TM1 were sufficient for interactions with the N terminally truncated receptor. In contrast, co-expression of the N terminally truncated receptor together with cell-associated or soluble EC region of the LH receptor did not allow ligand signaling. Unlike thrombin receptors, co-expression of the anchored EC region of the LH receptor together with the N-terminally truncated receptor did not allow ligand signaling despite moderate levels of human chorionic gonadotropin binding in transfected cells. These studies demonstrate that the co-expression of binding (+)/signaling (-) and binding (-)/signaling (+) receptor fragments partially restores ligand-induced signal generation and indicate the importance of TM1 of the LH receptor in the proper orientation of the EC ligand binding domain.
View details for Web of Science ID A1997XY97000046
View details for PubMedID 9312107
Receptors for the glycoprotein hormones, LH/CG, FSH, and TSH, are a unique subclass of the seven-transmembrane, G protein-coupled proteins with a large N-terminal extracellular (ecto-) domain. Although ecto-domains of gonadotropin receptors confer ligand binding, expression of soluble binding proteins has been difficult. We fused the ecto-domains of LH or FSH receptors to the single-transmembrane domain of CD8 and found that hybrid proteins anchored on the cell surface retained high-affinity ligand binding. Inclusion of a junctional thrombin cleavage site in the hybrids allowed generation of soluble receptor fragments that interfered with gonadotropin binding to their receptors and blocked cAMP production stimulated by gonadotropins. Cross-linking analyses confirmed the formation of high molecular weight complexes between receptor ecto-domains and their specific ligands. A similar approach also generated a soluble TSH receptor fragment capable of blocking TSH-induced signal transduction. When administered to rats, the soluble FSH receptor fragment retarded testis growth and induced testis cell apoptosis. These findings demonstrate the feasibility of generating ligand-binding regions of glycoprotein hormone receptors to selectively neutralize actions of gonadotropins and TSH, thus allowing future design of novel contraceptives and management of different gonadal and thyroid dysfunction. The present study represents the first successful derivation of soluble, ligand-binding domains from glycoprotein hormone receptors as functional antagonists. Similar approaches could allow generation of ecto-domains of related receptors to neutralize actions of ligands or receptor antibodies and to facilitate structural-functional analysis.
View details for Web of Science ID A1997XY29500009
View details for PubMedID 9328348
View details for Web of Science ID 000073305602733
The common alpha subunit of glycoprotein hormones contains five disulfide bonds. Based on the published crystal structure, the assignments are 7-31, 59-87, 10-60, 28-82, and 32-84; the last three comprise the cystine knot, a structure also seen in a variety of growth factors. Previously, we demonstrated that the efficiency of secretion and the ability to form heterodimers by alpha subunits bearing single cysteine residue mutants in the cystine knot were significantly reduced. These results suggested that the cystine knot is critical for the intracellular integrity of the subunit. To assess if the presence of the free thiol affected the secretion kinetics, we constructed paired cysteine mutants of each disulfide bond of the alpha subunit. The secretion rate for these monomers was comparable with wild type except for the alpha-10-60 mutant, which was 40% lower. The recovery of the alpha7-31 and alpha59-87 mutants was greater than 95%, whereas for the cystine knot mutants, it was 20-40%. Co-expression of the wild-type chorionic gonadotropin beta subunit with double cysteine mutants did not enhance the recovery of alpha mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the alpha10-60 or alpha32-84 mutants was less than 5%. Because subunit assembly is required for biological activity, studies on the role of these disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the alpha and beta subunits are genetically fused. The recovery of secreted tethered gonadotropins bearing mutations in the cystine knot was increased significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain alpha10-60 and alpha32-84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or signal transduction.
View details for Web of Science ID A1997XL73500032
View details for PubMedID 9218442
Using the ovary as a model system for studying the hormonal regulation of apoptosis, recent studies have revealed that the survival of growing follicles is under the regulation of a complex array of hormones through endocrine, paracrine, autocrine, or juxtacrine mechanism in a development-dependent manner. More effort is needed, however, to identify tissue-specific factors required for the survival of ovarian somatic and germ cells at specific stage of development. New insights based on characterization of conserved apoptotic effectors, both extracellular and intracellular, have suggested that apoptosis in ovarian cells may be mediated by apoptotic programs common to other cells but using specific members of the death domain proteins as well as ced-9/Bcl-2 and ced-3/ICE caspase families of genes. Future studies may provide new therapeutic modalities for different ovarian diseases caused by aberrant regulation of apoptosis in ovarian cells, including premature ovarian failure and polycystic ovarian syndrome. (Trends Endocrinol Metab 1997;8:207-213). (c) 1997, Elsevier Science Inc.
View details for Web of Science ID A1997XN65100006
View details for PubMedID 18406807
Glycoprotein hormones are noncovalent heterodimers comprised of a common alpha subunit and a hormone-specific beta subunit. Secretion and biologic action of these hormones are dependent on the formation of the heterodimer. The human LH beta subunit is unique among the other beta subunits in that it assembles inefficiently with the alpha subunit. To bypass this rate-limiting step, we constructed the LH single chains where the carboxy terminus of beta was fused to the amino terminus of alpha subunit through a linker. Compared to the human LH heterodimer, the extent of secretion was greater for the tethers although the rate was dependent on the nature of the linker. The LH single chains were biologically active even though there was loss of recognition by a LH-specific monoclonal antibody. This suggests that receptor binding of the single chains is not impaired by changes in the heterodimeric configuration resulting from tethering the subunits. In addition, single chains exhibited a remarkably greater in vitro stability than the heterodimer, implying that these analogs will be useful as diagnostic reagents and that their purification will be facilitated.
View details for Web of Science ID A1997XH58300031
View details for PubMedID 9219270
Progression of preantral follicle development is essential to further follicle maturation and ovulation, but there are few models for studying the regulation of preantral follicle survival and growth. We have evaluated preantral follicle survival in vivo and in vitro, and have developed a serum-free rat follicle culture system that can be used to characterize the regulation of preantral follicle growth and differentiation. Analysis of ovarian cell DNA fragmentation during the first wave of follicle growth in the infantile rat indicated negligible apoptosis up to day 16 of age. However, a major increase in apoptosis was found by day 18, a time point associated with the appearance of large antral follicles. In situ analysis confirmed that apoptotic DNA fragments were limited to antral follicles. Culture of individual preantral follicles mechanically dissected from ovaries of 12- or 14-day-old rats in serum-free conditions led to major increases in follicle cell apoptosis, similar to that seen in cultures of antral and preovulatory follicles. In contrast to antral and preovulatory follicles, treatment of preantral follicles with gonadotropins or cAMP analogs did not prevent apoptosis. However, treatment with 8-bromo-cGMP or 10% serum suppressed apoptosis by 75% in cultured preantral follicles. In situ analysis identified granulosa cells as the cell type susceptible to apoptosis regulation. Taking advantage of the ability of the cGMP analog to suppress apoptosis, we evaluated the potential of FSH as a growth factor. In the absence of serum, FSH treatment for 48 h did not affect follicle size compared to controls; however, treatment with the cGMP analog together with FSH increased follicle diameter (13%; P < 0.01) and viable cells (2.4-fold; P < 0.01) compared to control values. Immunoblot analysis further indicated that the inhibin-alpha content of the cultured follicles was increased by treatment with the combination of FSH and 8-bromo-cGMP, demonstrating the induction of follicle cell differentiation during culture. Therefore, we demonstrated that activation of the cGMP pathway promotes the survival of cultured preantral follicles and that in the presence of alpha cGMP analog, FSH is a growth and differentiation factor for preantral follicles. The present serum-free follicle culture model system will be useful in further evaluation of the regulation of growth and differentiation of preantral follicles.
View details for Web of Science ID A1997XA02500030
View details for PubMedID 9165031
Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.
View details for Web of Science ID A1997WV69100007
View details for PubMedID 9160709
Disrupting disulfide loops in the human chorionic gonadotropin beta subunit (CGbeta) inhibits combination with the alpha subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CGbeta subunits bearing paired cysteine mutations to a wild-type alpha (WTalpha) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CGbeta monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro. The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction. Moreover, these studies demonstrate that the single-chain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.
View details for Web of Science ID A1997WN14700002
View details for PubMedID 9054363
The majority of ovarian follicles undergo atresia, a hormonally controlled apoptotic process. Monitoring apoptotic DNA fragmentation provides a quantitative and sensitive endpoint to study the hormonal regulation of atresia in ovarian follicles. During follicle development, gonadotropins, together with local ovarian growth factors (IGF-I, EGF/TGF-alpha, basic FGF) and cytokine (interleukin-1 beta), as well as estrogens, activate different intracellular pathways to rescue follicles from apoptotic demise. In contrast, TNF-alpha, Fas ligand, presumably acting through receptors with a death domain, and androgens are atretogenic factors. These diverse hormonal signals probably converge on selective intracellular pathways (including genes of the bcl-2 and ICE families) to regulate apoptosis. With a constant loss of follicles from the original stockpile, the ovary provides a unique model for studying the hormonal regulation of apoptosis.
View details for Web of Science ID A1997WM92200016
View details for PubMedID 9074768
View details for Web of Science ID A1997BJ51H00001
View details for Web of Science ID A1997BJ51H00017
The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common alpha subunit non-covalently linked to a hormone-specific beta subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications and signal transduction. Structure-function studies of FSH and the other glycoprotein hormones are often hampered by mutagenesis induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH beta subunit to the amino end of the alpha subunit in the presence or absence of a natural linker sequence. In the absence of the CTP linker, the secretion rate was decreased over three fold. (The CTP sequence is the last 28 amino acids of the CG beta sequence and contains four serine-linked oligosaccharides). Unexpectedly however receptor binding/signal transduction was unaffected by absence of the linker. Molecular modelling of the tethers lacking the linker sequence show that the alignment of the alpha/beta domains in the single chain differ substantially from that seen in the heterodimer. These data show that the single chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.
View details for Web of Science ID A1996WC92300008
View details for PubMedID 9027345
We have generated transgenic mice overexpressing Bcl-2, an apoptosis suppression protein, in ovarian cells using the inhibit-alpha gene promoter/enhancer. Ovarian apoptotic DNA fragmentation induced in immature animals by a low dose (2 IU) of PMSG was suppressed by greater than 55% in transgenic mice compared to their wild-type littermates. Morphological and in situ DNA end-labeling analyses showed that granulosa cells in large antral follicles of wild-type animals undergo apoptosis, but most follicles in transgenic animals are healthy. When the animals were treated with a high dose (4 IU) of PMSG to stimulate follicular growth, spontaneous ovulation was observed in 14 of 23 (61%) of the transgenic animals, but in only 3 of 18 (17%) of wild-type siblings. Furthermore, transgenic females had a larger litter size (9.07 +/- 0.25 pups/litter; n = 29) than wild-type controls (7.54 +/- 0.26 pups/litter; n = 28; P < 0.01). These data suggested that decreased ovarian apoptosis in transgenic animals could lead to enhanced folliculogenesis and ovulatory potential. Moreover, aging transgenic mice are susceptible to the development of benign cystic ovarian teratoma (4 in 20 transgenic animals and 0 in 26 wild-type controls). Some tumor tissues showed respiratory and intestinal cell types, whereas others showed the development of central nervous system-like structures. Because the bcl-2 transgene in these animals is overexpressed in somatic cells, but not oocytes, these findings suggest that enhanced survival of selected somatic cells in transgenic mice could lead to germ cell tumorigenesis. Thus, overexpression of Bcl-2 protein in the ovary leads to decreased ovarian somatic cell apoptosis, enhanced folliculogenesis, and increased susceptibility to ovarian germ cell tumorigenesis in transgenic animals. The present mouse model allows future studies on intracellular signal pathways regulating follicular atresia and on the potential role of ovarian somatic cell factors in germ cell tumorigenesis.
View details for Web of Science ID A1996VN80900042
View details for PubMedID 8895354
Atretic demise rather than ovulation is the ultimate fate of the vast majority of ovarian follicles in mammals, affecting 70-99.9% of the follicles in various species. Recent studies have established that atretic degeneration of follicles is an apoptotic process, heralded by endonuclease degradation of DNA at internucleosomal sites, which generates DNA fragments in size multiples of 185-200 bp that are seen as distinct ladder bands after agarose gel electrophoresis. Using the well-characterized model of inducing atresia of preovulatory follicles in vivo by hypophysectomy and analyzing DNA fragmentation by autoradiography of size-fractionated DNA labeled at the 3' ends by [12P] dideoxy-ATP, we have examined the timing of atretic changes. DNA degradation was related to morphological signs of atresia, ovulability, and changes in follicular steroidogenesis. Rats were hypophysectomized on the morning of the day of proestrus, after which largest follicles were collected at various times. DNA fragmentation was analyzed in groups of five follicles. The increase in DNA fragments of low molecular weight up to 4 h after hypophysectomy was negligible (101 +/- 10%; 0 h time = 100%) but progressed 8, 12, 24, 48, and 72 h after hypophysectomy (143 +/- 20%, 168 +/- 27%, 235 +/- 29%, 3299 +/- 1075%, and 2249 +/- 805%, respectively; p < 0.03, n = 5). At 48 and 72 h, the extent of DNA degradation was higher than that observed in follicles cultured in a serum-free medium for 24 h. Likewise, staining of DNA by 4',6-diamido-2-phenylindole hydrochloride revealed apoptotic nuclei at 8 h after hypophysectomy (p < 0.01), and the percentage of such nuclei progressively increased afterwards. Thus, the increase in DNA fragmentation appeared concomitantly with atretic changes observed in previous studies (a decrease in ovulability at 6 h, and a spontaneous increase in progesterone accumulation and decrease in androgen and estrogen in follicles explanted 6 h after hypophysectomy) and preceded atresia detectable by morphological changes at 24 h. Detection of internucleosomal DNA degradation in preovulatory follicles early in the atretic cascade underscores the central role of apoptosis in ovarian follicle atresia.
View details for Web of Science ID A1996VN48800019
View details for PubMedID 8902220
In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.
View details for Web of Science ID A1996VN80900046
View details for PubMedID 8895358
The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.
View details for Web of Science ID A1996VL19700015
View details for PubMedID 8812137
Gonadotropin receptors are members of the seven-transmembrane (TM) receptor family. Several point mutations in TM V and VI and the intracellular loop 3 (i3) have been identified in the luteinizing hormone (LH) receptor gene, leading to constitutive activation of the receptor. Because gonadotropin receptors are highly conserved, we mutated the follicle-stimulating hormone (FSH) receptor at the corresponding amino acids. However, the FSH receptor mutants showed minimal increases in basal cAMP production. Taking advantage of this difference between the two receptors, we designed chimeric receptors with or without a point mutation in the i3 to identify the region in the LH receptor important for its constitutive activation. Introduction of the point mutation into chimeric receptors containing only TM V to VI from the LH receptor led to major increases in ligand-independent cAMP production. Furthermore, a chimeric receptor with only TM V and VI derived from the LH receptor can be rendered constitutively active by the mutation in the i3 from the FSH receptor. These results suggest that interactions between TM V and VI of the FSH receptor are essential for maintaining the receptor in the more constrained state, whereas interactions between these domains of the LH receptor are permissive for constitutively activating mutations in the i3.
View details for Web of Science ID A1996VG67200031
View details for PubMedID 8798412
Gonadotropin and TSH receptors represent a subgroup of seven transmembrane-spanning, G protein-coupled receptors with a large extracellular ligand-binding region. After ligand binding to their receptors, the majority of actions of gonadotropins and TSH are believed to be mediated by the cAMP-protein kinase A pathway. Although formation of inositol phosphates (IP) has been reported after stimulation of rodent gonadotropin receptors, activation of phospholipase C after ligand binding of human LH or FSH receptors has not been investigated. Human gonadotropin receptors were transiently expressed in 293 cells, and the agonist-induced stimulation of IP formation was measured. The LH receptor responded to a saturating dose of human CG (hCG) with a 5.2-fold increase of IPs whereas the FSH receptor responded to a saturating dose of FSH with only a 50% increase. On the basis of these differences and in view of the homologous nature of the two gonadotropin receptors, chimeric receptors were constructed using domain transfer to identify the regions in the human LH receptor important for phosphatidylinositol hydrolysis. Chimeric receptors containing the entire extracellular region of the FSH receptor and the seven transmembrane region plus the cytoplasmic tail of the LH receptor responded to FSH treatment with a 4.7-fold increase in IP accumulation. In contrast, the chimeric receptor with the extracellular region of the LH receptor and the TM region plus the cytoplasmic tail of the FSH receptor responded minimally (50%) to hCG treatment. When the C-terminal third (from TM V to the cytoplasmic tail) of the FSH receptor was replaced with the LH receptor sequence, the chimeric receptor still responded to FSH treatment with a large (6.2-fold) increase in IP release, similar to that of the wild type LH receptor (to hCG), suggesting that C-terminal third of the human LH receptor confers IP signaling ability. This functional domain was further divided into two areas, namely TM V to TM VI and TM VII to the cytoplasmic tail. The chimeric receptors F(I-IV)L(V-VI)F(VII-C)R and F(I-VI)L-VII-C)R, in which these two regions of the FSH receptor were replaced by the corresponding sequences of the LH receptor, responded to FSH treatment with partial increases in phosphatidylinositol hydrolysis (2.0- and 3.7-fold, respectively). Furthermore, when TM VII and the cytoplasmic tail of the LH receptor were replaced with the corresponding sequence of the FSH receptor, this chimeric receptor showed a diminished (2.0-fold) response to hCG in IP release. For all the chimeric receptor constructs analyzed, overall expression, equilibrium binding constants, and adenyl cyclase activation were not altered. Thus, unlike studies using chimeric muscarinic and dopaminergic receptors in which the second and third intracellular loops were found to be important for IP signaling, the entire C-terminal third of the human LH receptor is important for IP release. Future analysis using the chimeric receptor approach should provide new information on the structure-function relationship of gonadotropin, TSH, and other seven transmembrane-spanning receptors.
View details for Web of Science ID A1996VF48200008
View details for PubMedID 8885247
Male-limited precocious puberty (MPP) is a gonadotropin-independent disorder that occurs sporadically or is inherited in an autosomal dominant, male-limited pattern. Recent studies have identified constitutively activating missense mutations in the human luteinizing hormone receptor (hLHR) gene leading to Leydig cell activation and precocious puberty. Patients with sporadic MPP (SMPP) or with different ethnic backgrounds appear to have a greater likelihood of having novel mutations. In the current study we examined genomic DNA from two unrelated cases of SMPP of African-American descent for novel mutations of the hLHR gene. A heterozygous A to C transversion at nucleotide 1723 resulting in substitution of Leu for lle575 in transmembrane helix 6 was identified. Human embryonic kidney cells transfected with cDNA for the mutant hLHR-I575L, created by polymerase chain reaction-based mutagenesis of the wild-type (hLHR-wt) cDNA, exhibited increased basal levels of cAMP production in the absence of agonist, indicating constitutive activation. Surface expression of hLHR-I575L, as reflected by human chorionic gonadotropin binding, was diminished compared to hLHR-wt, while agonist affinity was unaffected. With the exception of two polymorphic bases, no mutation was identified within the coding sequence of the hLHR in the second case of SMPP. We conclude that I575L is a unique constitutively activating mutation that impairs cell surface expression of the receptor but does not alter agonist affinity. Furthermore, mutations of the hLHR gene causing SMPP are highly heterogeneous and may be found in regions other than exon 11 of the hLHR. Last, patients with MPP from different ethnic backgrounds are likely to have novel mutations.
View details for Web of Science ID A1996VB36100008
View details for PubMedID 8812739
The human LH receptor (hLHR) is a member of the G protein-coupled receptors characterized by the presence of seven-transmembrane (TM) helices. Inactivating mutations of the hLHR lead to Leydig cell hypoplasia (LCH), a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cell differentiation. We have identified three mutations of the hLHR in a patient with LCH: deletion of exon 8 (delta Exon 8), A872G transition resulting in Asn291Ser substitution in the extracellular domain, and C1847A transversion resulting in Ser616Tyr substitution in the seventh TM helix. Nucleotide sequencing, gene dosage, and allele-specific amplification analyses revealed that exon 8 deletion and the two missense mutations are present in different alleles of the hLHR. Constructs of mutated hLHR (hLHR-delta Exon8, hLHR-872/1847, hLHR-1847, and hLHR-872) were used to transfect 293 cells, and the properties of the hLHR expressed were examined. Ligand-binding assays failed to detect the expression of hLHR-delta Exon8. Transfectants expressing hLHR-872/1847 demonstrated greatly reduced ligand binding and ligand-induced cAMP accumulation in comparison to those expressing wild type hLHR. Similar reduction in cAMP accumulation was observed in transfectants expressing hLHR-1847, but not hLHR-872 alone. These findings suggest that, in addition to the 7-TM helices, the polypeptide encoded by exon 8 plays an important role in LHR expression and signal transduction. On the other hand, glycosylation of Asn291 may not be critical for these activities. These results also establish that LCH can result from impaired signal transduction due to compound heterozygous mutations. Implications of these mutations on structure-function relationship of the hLHR and the genotype-phenotype correlation in LCH are discussed.
View details for Web of Science ID A1996VA65100008
View details for PubMedID 8843415
Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.
View details for Web of Science ID A1996UB89100042
View details for PubMedID 8625923
Apoptosis is an important cellular process by which superfluous or unwanted cells are deleted from an organism during tissue remodeling and differentiation. Recent studies have demonstrated the role of this programmed cell death or "controlled cell suicide" in the physiological function of an organism. Suppression of apoptosis increases the susceptibility of an individual to malignancy whereas uncontrolled cell death is associated with degenerative diseases. Normal development of both female and male gonads is characterized by massive cell death. More than 99% of ovarian follicles endowed at early life are destined to undergo apoptosis and the exhaustion of these follicles serves as a "clock" for female reproductive senescence. In the testis, up to 75% of male germ cells also undergo apoptosis, perhaps as a mechanism to delete superfluous or defective germ cells. Gonadal cell apoptosis provides valuable models to study hormonal regulation of apoptosis. In the ovary, gonadotropins, estrogens, growth hormone, growth factors (IGFI, EGF/TGF-alpha, basic FGF), cytokine (interleukin-1 beta) and nitric oxide act in concert to ensure the survival of preovulatory follicles. In contrast, androgens, interleukin-6 and gonadal GnRH-like peptide are apoptotic factors. Developmental studies further indicate that fractions of endowed follicles are recruited throughout the reproductive life whereas most of the primordial follicles are "arrested" at the initial stage of development for a prolonged time. Because a transcriptional factor WT1 is expressed in high levels in follicles at early stages of development and because WT1 over-expression represses the promoter activity of inhibin-alpha gene, this nuclear protein may be important in the maintenance of follicles at early stages of development. Once a cohort of follicles is recruited to grow, it is destined to undergo apoptosis unless rescued by survival factors. After puberty onset and under gonadotropin stimulation, some of the growing antral follicles are "selected" to continue their final maturation and secrete high levels of estrogens to trigger ovulation. Following repeated cycles of recruitment, atresia or ovulation, the follicle reserve is exhausted, thus signaling the onset of reproductive senescence. Although the somatic granulosa cell is the major cell type undergoing apoptosis in the ovary, the germ cells in the testis also exhibit signs of apoptotic cell demise. In the testis, gonadotropins and androgens act as survival factors whereas exposure to elevated temperature in cryptorchid testes increases apoptosis. In the seasonally breeding hamster model, photoperiod-entrained regression and recrudescence of testis tissue serves as a unique natural model of apoptosis. With recent advances in our understanding of the cellular mechanism of apoptosis, including the elucidation of the Ced9/bc12 and Ced3/ICE family of proteins, further investigation of gonadal apoptosis may lead to a better understanding of gonadal degenerative disorders (such as premature ovarian failure and oligospermia), reproductive senescence and tumorigenesis. The gonadal model should also be valuable in studying the regulation of intracellular apoptosis genes by external hormonal signals.
View details for Web of Science ID A1996BJ31P00017
View details for PubMedID 8701090
View details for Web of Science ID A1996BG49C00006
View details for Web of Science ID A1996BG49C00012
The inhibin-alpha gene is expressed in a tissue-specific manner, and its protein product dimerizes with one of two beta-subunits to form bioactive heterodimers. To characterize the cis-acting elements involved in directing gonad- and adrenal-specific expression of inhibin-alpha, transgenic mice were generated that carried 2.5 or 6 kilobases (kb) of the 5'-flanking region of the mouse inhibin-alpha gene driving the human bcl-2 complementary DNA. Using an antibody specific for human Bcl-2, Western blotting and immunocytochemical analyses showed that both enhancer/promoter fragments direct transgene expression to the ovary, testis, and adrenal gland. The 6-kb fragment targeted the ovarian transgene expression in interstitial cells and young corpora lutea as well as granulosa and thecal cells of secondary, antral, and preovulatory follicles. In ovaries of animals with the 2.5-kb fragment, transgene expression was also detected in interstitial cells and young corpora lutea, but only in granulosa and thecal cells from antral and preovulatory follicles. The ovarian transgene expression in animals carrying the 6-kb inhibin-alpha promoter/bcl-2 construct was stimulated by gonadotropin treatment, with greater than 10-fold increases observed 2 days after PMSG stimulation. In the testes of both types of transgenic animals, immunoreactive Bcl-2 was predominantly detected in Sertoli cells of seminiferous tubules. Sporadic expression was also observed in some interstitial cells. In the adrenal gland, reporter protein was detected in the zona fasciculata of both types of transgenic animals during adult life; however, transgene expression was detected in zona fasciculata of young (21-day-old) animals with the 6-kb, but not the 2.5-kb, promoter construct. Thus, the 2.5-kb inhibin-alpha 5'-proximal DNA sequence directs transgene expression in mature ovarian follicles and testicular Sertoli cells. In contrast, enhancer elements in the 6-kb fragment are required for expression in preantral follicles and in the adrenal of immature animals. The inhibin-alpha promoter/enhancer used here represents unique DNA sequences for ovarian-specific transgene expression and is useful for future analysis of gonadal and adrenal cell functions.
View details for Web of Science ID A1995TH00800045
View details for PubMedID 7588311
WT1, a gene deleted in some Wilms' tumors, encodes a transcription factor with zinc fingers and shares homology with proteins in the early growth response gene family. Although defects in the WT1 gene are associated with nephroblastoma and genitourinary malformation, the specific function of WT1 in the gonads remains unclear. We investigated the expression of WT1 transcripts in rat ovary during follicle development by Northern blotting, RNase protection assay, and in situ hybridization. Abundant WT1 transcripts were found in the ovary, testis, uterus, and kidney, with lower levels in the heart and pancreas. Treatment with estrogen or gonadotropins did not affect the concentration of ovarian WT1 mRNA. In situ hybridization analysis indicated that ovarian WT1 mRNA is expressed exclusively in the surface epithelium and granulosa cells of primordial, primary, and secondary follicles, and its levels decrease during follicle growth. Although RNase protection assay suggested the presence of four alternatively spliced forms of WT1 mRNA, the ratio of these transcripts remains constant during ovarian growth. Developmental changes in the expression of two granulosa cell differentiation marker genes, inhibin-alpha and FSH receptor, were found to be inversely correlated with WT1 levels. Because potential WT1-binding sites were found in the promoter of inhibin-alpha gene, we further tested whether WT1 might regulate the expression of this gene. Cotransfection of a WT1 expression vector with a promoter reporter plasmid of inhibin-alpha resulted in the repression of promoter activities in CHO cells in a dose-dependent manner. These results suggest that WT1 is expressed in high levels in granulosa cells of primordial, primary, and secondary follicles but decreases with follicle development. This transcription factor might be a repressor of ovarian differentiation genes in the granulosa cells and play a role in arresting the differentiation of immature follicles.
View details for Web of Science ID A1995RY96100010
View details for PubMedID 8544844
View details for Web of Science ID A1995RW68701250
The present world population of 5.6 billion is projected to reach 9 billion by the year 2025. Overpopulation remains one of the overwhelming issues of the 21st century, but only limited effort and resources have been allocated to designing new contraceptives, as evidenced by the diminished interest of the pharmaceutical industry and funding agencies. Major advances have been made recently in our understanding of the genetic basis of an individual's risk to various reproductive cancers and sex steroid-related diseases. It has also become apparent that agonistic and antagonistic analogues of sex steroids with tissue-specific actions can be formulated. These new insights provide the opportunity to develop the next generation of 'designer' contraceptive pills with disease-prevention benefits.
View details for Web of Science ID A1995RU03700014
View details for PubMedID 8567829
Leydig cell hypoplasia (LCH) is a form of male pseudohermaphroditism in which Leydig cell differentiation and testosterone production are impaired. This report describes the first case of a nonsense mutation (A1635C) in exon 11 of the human luteinizing hormone receptor (hLHR) gene in two sisters with LCH. This mutation causes loss of function of the receptor by introducing a stop codon at residue 545 in transmembrane helix 5 of the hLHR. Surface expression of the truncated hLHR (hLHR-t545) in human embryonic kidney cells stably transfected with cDNA encoding hLHR-t545 was diminished compared to the wild-type hLHR and hCG-induced cAMP accumulation was impaired. These results establish that single base mutations in exon 11 of the hLHR gene can produce inactivation as well as activation of the hLHR. Furthermore, they demonstrate that functional domains between transmembrane helix 5 and the C-terminal cytoplasmic tail of the hLHR are required for normal cell surface expression of the receptor and signal transduction.
View details for Web of Science ID A1995RM34000025
View details for PubMedID 7581384
A growing body of evidence suggests that intraovarian interleukin-1 beta (IL-1 beta) may play an intermediary role in the ovulatory process. Furthermore, induction of nitric oxide (NO) by IL-1 beta has been reported in a wide variety of tissues. As the majority of ovarian follicles undergo an atretic degeneration process involving apoptotic cell death, we set out to determine whether IL-1 beta rescues follicles from apoptosis and the possible involvement of NO. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends with [32P]dideoxy-ATP. Follicular NO production was also determined by a colorimetric method. Treatment with IL-1 beta dose-dependently suppressed the spontaneous onset of apoptosis in cultured follicles, but stimulated NO production. In contrast, the addition of IL-1 receptor antagonist eliminated both effects of IL-1 beta, confirming receptor mediation. Follicles treated with sodium nitroprusside, a NO generator or an analog of cGMP, the second messenger for NO, also showed decreased follicle apoptosis. Moreover, the addition of NG-monomethyl-L-arginine, a NO synthase inhibitor, reversed both IL-1 beta stimulation of NO production and suppression of apoptosis, suggesting a mediatory role of NO in these IL-1 beta effects. Gonadotropins also prevent follicle apoptosis. Of interest, treatment with hCG stimulated NO production, and the hCG suppression of follicle apoptosis and stimulation of NO production were partially blocked by cotreatment with IL-1 receptor antagonist, indicating the mediation of endogenous IL-1 beta. Treatment with IL-1 beta also stimulated a small increase in the production of cAMP, estrogen, and progesterone. Taken together, these findings suggest that IL-1 beta is a survival factor for ovarian follicles, and its action is partially mediated via NO and cGMP generation. Moreover, part of the suppressive action of gonadotropins on follicle apoptosis is mediated by endogenously produced IL-1 beta.
View details for Web of Science ID A1995RE89700044
View details for PubMedID 7540548
A growing body of evidence suggests that growth hormone (GH) plays a role in regulating ovarian function by augmenting gonadotropin stimulation of granulosa cell differentiation and folliculogenesis. The majority of follicles in the mammalian ovary do not ovulate, but instead undergo a degenerative process (atresia) involving apoptotic cell death. The objective of the present study was to investigate the role of GH in regulating follicle apoptosis and to determine whether or not insulin-like growth factor-I (IGF-I) mediates GH action in this process. Preovulatory follicles obtained from eCG-primed rats were cultured for 24 h in serum-free conditions with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3' ends with [32P]dideoxy-ATP. Culture of preovulatory follicles resulted in a spontaneous onset of apoptotic DNA fragmentation that was suppressed by ovine GH (oGH) in a dose-dependent manner, reaching a maximum of 65% suppression. To rule out the effect of residual gonadotropin in the oGH preparation, follicles were also cultured with recombinant bovine growth hormone (rbGH). Like oGH, rbGH suppressed apoptotic DNA fragmentation. Our earlier study indicated that hCG and FSH treatment also suppress apoptosis in the present model system, but no additive effect of GH and either hCG or FSH on the suppression of apoptosis was observed. To determine whether the observed effect of GH action on follicle apoptosis is mediated by IGF-I, three types of studies were carried out.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1995RC85000003
View details for PubMedID 7545438
Disturbed human reproductive function may be caused by environmental and/or genetic factors. Much information related to single gene defects underlying reproductive failure has become available in recent years due to advances in molecular biology. In this review, techniques currently applied for deoxyribonucleic acid (DNA) analysis are addressed. We also highlight underlying molecular mechanisms and the corresponding clinical presentation of single gene defects affecting (i) the hypothalamic-pituitary-gonadal axis, resulting in disturbed gonadotrophin-releasing hormone (GnRH) neuron migration, or leading to defective gonadotrophins, gonadotrophin receptors and the Gs alpha protein; (ii) gonadal and adrenal steroid biosynthesis and (iii) steroid and insulin receptors. The potential genetic basis of polycystic ovary syndrome is also discussed. Although disease states caused by well-defined genetic abnormalities appear to represent only a small proportion of those found in the patient population, it should be considered that these affected individuals represent only the most severe cases in a wide spectrum of genetic abnormalities underlying disturbed fertility. Comprehension of these extreme cases will provide the basis for the elucidation of more common reproductive disorders as the result of subtle genetic changes or increased susceptibility to environmental factors.
View details for Web of Science ID A1995QX12200019
View details for PubMedID 7650129
In the present study, we examined the expression of LH and CG receptor messenger RNA (mRNA) in human corpora lutea (CL) during the menstrual cycle and pregnancy. Poly(A)-enriched RNA was extracted from CL and analyzed by Northern and slot blots, using a radiolabeled complementary RNA probe derived from the human LH receptor complementary DNA. Northern blot analysis indicated the presence of multiple LH receptor mRNA transcripts with molecular sizes of 8.0, 7.0 and 4.5 kilobases in human CL during the menstrual cycle. The predominant transcript was 4.5 kilobases in size. However, no hybridization signals were observed in nongonadal tissues (heart, liver, and kidney). Densitometric analyses revealed that the levels of LH receptor mRNA increased from early luteal phase to midluteal phase and subsequently decreased during late luteal phase. After the onset of menstruation, the LH receptor mRNA level was undetectable in the regressing CL. Moreover, radioligand receptor assay (RRA) showed a close parallelism between LH receptor mRNA levels and LH receptor content in CL throughout the menstrual cycle. LH receptor mRNA expression was also found in CL during early pregnancy. The level of LH receptor mRNA was relatively high in early pregnancy CL, whereas LH receptor content was low. Using in situ hybridization, LH receptor mRNAs were uniformly expressed in both large and small luteal cells during early and midluteal phase and early pregnancy, but not in regressing CL. In conclusion, these data demonstrate that the regulation of LH receptor content in human CL during luteal phase is associated with similar changes in the receptor message levels, suggesting the physiological roles for LH receptor mRNA during the menstrual cycle in the human. In addition, the expression of LH receptor mRNA was demonstrated in human CL during early pregnancy.
View details for Web of Science ID A1995QR24300071
View details for PubMedID 7714122
One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a hormone-specific beta subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide hCG molecule not only was efficiently secreted but also displayed an increased biological activity in vitro and in vivo. These data show that the alpha and hCG beta subunits encoded as a single chain retain a biologically active conformation similar to that seen in the heterodimer. This approach can be used to investigate structure-function relationships of the glycoprotein hormone family that were previously not tractable because of the absolute dependence on assembly for the biological response. Moreover, other bioactive multisubunit ligands can be engineered where the combination efficiency and specificity of heterodimers and homodimers are otherwise difficult to control.
View details for Web of Science ID A1995QM40800053
View details for PubMedID 7892221
View details for PubMedCentralID PMC42419
Genomic DNA from 32 unrelated families with male-limited precocious puberty was examined for the previously described Asp-578-->Gly, Met-571-->Ile, and Thr-577-->Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR). Twenty-eight families had the inherited form of the disorder, and of these, 24 were found to have the Asp-578-->Gly mutation. Four additional mutations were found among the remaining four families with the inherited form and in four sporadic cases of the disorder: an A-->C transversion resulting in substitution of leucine for Ile-542 in the fifth transmembrane helix, an A-->G transition resulting in substitution of glycine for Asp-564 in the third cytoplasmic loop, a G-->T transversion resulting in substitution of tyrosine for Asp-578 in the sixth transmembrane helix, and a T-->C transition resulting in substitution of arginine for Cys-581 in the sixth transmembrane helix. Human embryonic kidney cells transfected with cDNAs for each of the mutant hLHRs, created by PCR-based mutagenesis of the wild-type hLHR cDNA, exhibited increased levels of basal cAMP production in the absence of agonist, indicating constitutive activation of the mutation hLHRs. Three of the additional mutations had specific features: Ile-542-->Leu and Cys-581-->Arg appeared ligand-unresponsive, whereas Asp-578-->Tyr appeared to correlate genotype with phenotype. We conclude that the region spanning nt 1624-1741 of exon 11 is a hotspot for heterogeneous point mutations that constitutively activate the hLHR and cause male-limited precocious puberty.
View details for Web of Science ID A1995QM40800025
View details for PubMedID 7892197
View details for PubMedCentralID PMC42391
Recent studies have demonstrated apoptotic DNA fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis apoptotic DNA fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two injections at 2-day intervals). Pretreatment with the GnRH antagonist increased apoptotic DNA fragmentation in rats between 16-32 days of age but not in younger and adult animals demonstrating an age-related change in gonadotropin dependence. To identify the exact testis cell type undergoing apoptosis, in situ analysis of DNA fragmentation was performed. In rats at 16-24 days of age, spermatocytes in selected tubules were found to have increased DNA fragmentation. In contrast, neither Leydig cells nor Sertoli cells were affected. In 32-day-old and adult animals, increased DNA fragmentation was seen in early primary spermatocytes of some tubules. Treatment with GnRH antagonist increased the number of cells with DNA fragmentation as well as percentage of tubules affected. In animals between 16-32 days of age, meiotic spermatocytes were labeled, whereas early spermatids were also labeled in 24- and 32-day-old animals. In adult animals, the level of apoptotic DNA fragmentation was not affected by GnRH antagonist treatment. However, DNA isolated from specific stages of the seminiferous tubules of adult animals showed stage-specific changes of apoptotic DNA fragmentation with 2-fold higher levels found in stages I and XII-XIV compared with stage VIII. In situ analysis of adult testis demonstrated that spermatocytes were the major cell type affected. In conclusion, the present study demonstrated that at least three factors determine the onset of apoptosis of the male germ cells: 1) the developmental stage of the animal; 2) serum levels of gonadotropins, especially FSH; and 3) specific stage of the seminiferous epithelial cycle. The present approach provides the basis for future analysis of the role of gonadotropins and other factors in the regulation of testis cell degeneration in normal and pathological states.
View details for Web of Science ID A1995QD58400002
View details for PubMedID 7828558
View details for Web of Science ID A1995RD90200372
The hCG beta-subunit contains a carboxy-terminal extension bearing four serine-linked oligosaccharides [carboxy-terminal peptide (CTP)], which is important for maintaining its longer half-life compared with the other glycoprotein hormones. Previously, we enhanced the in vivo half-life of FSH by fusing the CTP to the carboxy end of FSH beta coding sequence. The alpha-subunit is common to the glycoprotein family. We constructed alpha-subunit CTP chimeras, since such analogs with the appropriate O-linked glycosylation and conformation would increase the in vivo stability of the entire glycoprotein hormone family. Two chimeras were constructed using overlapping polymerase chain reaction mutagenesis: a variant with CTP at the carboxy end and another analog with the CTP at the N-terminal region of the subunit, between amino acids 3 and 4. The latter design was based on models showing that the amino-terminal region of alpha is not involved in assembly with the beta-subunit, nor is it essential for receptor binding and signal transduction. These chimeras were cotransfected with the hCG beta gene into Chinese hamster ovary cells. The chimeras were secreted and combined efficiently with the CG beta-subunit, comparable to the wild type alpha-subunit. CG dimers containing the alpha-subunit chimera with CTP at the carboxy end of the subunit had a much lower binding affinity for the hLH-hCG receptor in vitro, whereas the binding of the dimer containing the CTP at the amino-terminal end of the subunit was similar to wild type hCG. Furthermore, the in vivo activity of this analog was enhanced significantly. Moreover, regardless of the two insertion points in the alpha-subunit, the CTP sequence was O-glycosylated. These data suggest that the entire signal for O-glycosylation is primarily contained within the CTP sequence and is not dependent on the flanking regions of the recipient protein. The transfer of CTP to the alpha-subunit of hCG results in an agonist with prolonged biological action in vivo. These data further support the rationale for using the CTP as a general target to increase the potency of bioactive glycoproteins.
View details for Web of Science ID A1995QF72500006
View details for PubMedID 7539107
View details for Web of Science ID A1995RD90200368
Reproductive activity in the Djungarian hamster is controlled by seasonal variations in day length. Exposure to long days stimulates testis development, while exposure to short days induces testis regression. We recently found that testis regression after gonadotropin deprivation in rats is associated with increases in apoptosis. Here we sought to determine whether or not apoptosis is associated with the testis regression and/or recrudescence that occurs naturally in seasonally breeding mammals. Newborn male hamsters were maintained on long days (16L:8D) until 3 wk of age before being transferred to short days (8L:16D). Following decreases in serum FSH within 3 days of exposure to short days, testis weight decreased by 52% at Day 10, reaching a 70% decrease after 21 days. Analysis of testis cell DNA fragmentation showed a 4.9-fold increase of low-molecular-weight DNA as early as 5 days after transfer to short days; this was followed by a time-dependent decrease. The observed increases in testis cell apoptosis were correlated with decreases in serum testosterone, but decreases in Leydig cell LH receptor content were delayed. In a second study, 6-wk-old hamsters with regressed testes due to a 3-wk exposure to short days were transferred back to long days. After increases in serum FSH within 3 days of photostimulation, a 2-fold elevation in testis weight was found at Day 5. The increase in testis weight was associated with a 65% decrease of testis apoptosis within 5 days of photostimulation. Also, increases in serum testosterone and LH receptor content were observed after 5 and 10 days of exposure to long days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994PR43000031
View details for PubMedID 7888511
Although the majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in apoptotic DNA fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on apoptotic DNA fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1994PR66700018
View details for PubMedID 7525255
Although surgical induction of cryptorchidism in the rat is known to cause infertility due to disruption of spermatogenesis, the exact cellular mechanism responsible for the degenerative changes in cryptorchid testes is unclear. Using a sensitive autoradiographic method for the detection of apoptotic DNA fragmentation, we have investigated the effect of experimentally induced cryptorchidism on apoptotic cell death in testes of immature rats. Bilateral or unilateral cryptorchidism decreased the weight of affected testes within 4 days; these decreases (24-27%) became significant (P < 0.05) at 7 days after the operation. Testes of sham-operated animals contained predominantly high molecular weight DNA (> 15 kb), whereas DNA cleavage into low molecular weight ladders characteristic of apoptosis was increased by induction of bilateral cryptorchidism in a time-dependent manner, i.e., 2.0-, 2.8-, and 4.2-fold (p < 0.05) at 2, 4, and 7 days after operation, respectively. In unilaterally cryptorchid animals, sham-operated testes also contained predominantly high molecular weight DNA, whereas induction of cryptorchidism of the contralateral testes increased DNA cleavage into low molecular weight fragments 3.0-, 2.8-, and 3.9-fold (p < 0.05) at 2, 4, and 7 days after the operation, respectively. In situ analysis of DNA fragmentation in testes of unilaterally cryptorchid rats at 7 days after the operation indicated that germ cells, mainly primary spermatocytes were affected and that the percentage of seminiferous tubules containing labeled cells increased in the operated testis as compared to the contralateral control in the same animal.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994PL95500006
View details for PubMedID 7849188
Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha subunit but differ in their hormone-specific beta subunits. Site-directed mutagenesis was used to examine the role of the five disulfide bonds in the alpha subunit on the folding, assembly with the hCG beta subunit, and in cases where dimer formation occurred, receptor binding and signal transduction. Cysteine residues in the disulfide bonds formed by cysteines 7-31, 10-60, 28-82, 59-87, and 32-84 (Lapthorn, A., Harris, D. Littlejohn, A., Lustbader, J. Canfield, R., Machin, K., Morgan, F., and Isaacs, N. (1994) Nature 369, 455-461) were converted to alanine, and these mutants were transfected alone or together with the wild-type hCG beta gene into Chinese hamster ovary cells. The alpha Cys-10, 28, 60, 82, and 84 mutants were not secreted and in most cases were degraded at a faster rate than the native subunit. In addition, these mutants failed to assemble with the hCG beta subunit. Mutants with alterations at alpha Cys-7, 31, 32, 59, or 87 were secreted and combined with the beta subunit. Heterodimers containing a 7-31 double mutant bound to human lutropin-chorionic gonadotropin receptor expressed in transfected human fetal kidney cells, and stimulated cAMP comparable to wild-type hCG. Dimers containing the beta subunit with either single mutant alpha 59, alpha 87, alpha 32, or the alpha 59-87 double mutant showed much lower affinity for the receptor than wild-type hCG. These results suggest that disulfide bonds associated with alpha 7, alpha 31, alpha 59, alpha 87, and alpha 32 are not essential for the alpha subunit to fold into a form that will combine with the hCG beta subunit and to produce a biologically active dimer. This contrasts with observations of the hCG beta subunit where all the disulfide bonds are required for efficient combination and folding (Suganuma, N., Matzuk, M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307). In addition, the lack of secretion of some mutants reflects previous observations that proteins which do not fold correctly are rapidly degraded. Thus, alpha subunit mutants which fold properly are secreted and can form heterodimers.
View details for Web of Science ID A1994PQ49100049
View details for PubMedID 7929256
A bacterial expression system for the beta-subunit of hCG (hCG beta) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG beta in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG beta complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG beta (rhCG beta) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG beta were recovered from 1 liter culture, rhCG beta was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCG alpha and the purified rhCG beta/urinary alpha dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG beta/urinary alpha dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG beta. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG beta are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG beta in bacteria and of folding it in vitro implies that the beta-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.
View details for Web of Science ID A1994PD46000015
View details for PubMedID 8070386
Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutants develop gonadal sex cord-stromal tumors. To investigate their endocrine and proliferative characteristics, gonadal tumor cells were maintained in vitro. Cells from inhibin-alpha-deficient mice multiplied poorly; however, cells from mice deficient in both inhibin-alpha and p53 proliferated rapidly and showed higher saturation density and plating efficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clonal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to exogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatment. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta A and beta B mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta A-subunit protein. We further tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replication in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follistatin action. Incorporation of thymidine into DNA of these cells was also stimulated by activin. In addition, treatment with antiactivin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by specific receptors because cross-linking of activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells. Our findings indicate that activin can act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-alpha and p53-deficient mice and inhibits progesterone production. These tumor cell lines are useful for studies on the regulation of gonadal cell proliferation and steroidogenesis as well as the signaling pathway mediating activin action.
View details for Web of Science ID A1994PB57600004
View details for PubMedID 7997239
View details for Web of Science ID A1994NH51600576
Pituitary gonadotropin FSH acts exclusively on ovarian granulosa cells by binding to specific plasma membrane receptors. Transforming growth factors alpha and beta (TGF alpha and TGF beta), produced locally within the ovary, have been shown to regulate diverse follicle functions, although their potential role in the regulation of FSH receptors has not been assessed. Our first objective was to demonstrate developmental changes in the expression of FSH receptor gene and protein; we then analyzed the regulation of FSH receptor expression by TGF beta s and TGF alpha in cultured granulosa cells. Analysis of steady-state FSH receptor mRNA and protein levels in neonatal and prepubertal ovaries revealed the existence of two predominant FSH receptor mRNA transcripts, 7.0 and 2.5 kb in size, showing a dramatic increase between Day 15 and Day 18 of age followed by a plateau up to 27 days of age. A close parallelism in the developmental changes in FSH receptor mRNA levels and FSH receptor content was observed. Cultured granulosa cells obtained from estrogen-treated immature rats exhibited FSH receptor transcripts similar in size to those seen in whole ovaries. Treatment of granulosa cells for 48 h with TGF beta 1 increased the levels of FSH receptor mRNA for both the 7.0- and 2.5-kb transcripts in a dose-dependent manner (ED50, 1.5 ng/ml), with a maximal 4.0 +/- 0.8-fold increase over control levels observed in response to 10 ng/ml TGF beta 1. Also, TGF beta 2 was as potent as TGF beta 1 in increasing FSH receptor mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994NB91100027
View details for PubMedID 8199274
View details for Web of Science ID A1994PL77100003
The majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death. Although GnRH and its agonists have been shown to suppress ovarian growth and differentiation in hypophysectomized rats, studies on the induction of follicle atresia by GnRH are contradictory. In the present study, the direct effect of GnRH on the occurrence of apoptosis in the ovary was investigated in hypophysectomized estrogen-treated immature rats. Starting 2 days after operation and estrogen capsule implantation, rats were treated with a GnRH agonist (GnRHa; [desGly10,D-Phe6,Pro9-N-ethylamide] GnRH; 50 micrograms/injection, twice daily). Total ovarian DNA was isolated 48 h later, labeled at the 3'ends with [32P]dideoxy ATP, and size-fractionated. Compared to that in control animals, treatment with GnRHa increased DNA fragmentation in multiples of 180 basepairs, a hallmark of apoptosis, demonstrating that GnRH directly increases ovarian apoptotic cell demise. In contrast, FSH treatment (10 micrograms/injection, twice daily) decreased apoptotic DNA fragmentation, and the antiapoptotic effect of FSH was partially blocked by concomitant treatment with GnRHa. The apoptosis-inducing effect of GnRHa was time and dose dependent, with a significant increase seen after 24 h of treatment and a maximal 5.5-fold increase with 10 micrograms GnRHa/injection. Similar to studies using DNA isolated from whole ovaries, DNA obtained from isolated granulosa cells also showed a time- and dose-dependent increase in DNA fragmentation after GnRHa treatment. The effect on DNA fragmentation by GnRHa was mediated by ovarian GnRH receptors, because a potent GnRH receptor blocker, Azaline B, prevented GnRHa action. In addition, in situ end labeling of DNA using digoxigenin-dideoxy-UTP showed that DNA fragmentation was confined to the granulosa cells of preantral and antral follicles. No GnRHa-induced apoptosis was detected in granulosa cells of primary follicles or in thecal and interstitial cells. These data suggest that GnRH directly increases apoptotic cell death in the ovary, and the GnRH action is confined to the granulosa cells. These data provide a basis for future studies on the mechanism of follicular atresia and the regulation of ovarian endonuclease by GnRH.
View details for Web of Science ID A1994MQ62100035
View details for PubMedID 8275940
View details for Web of Science ID A1994BA41H00059
View details for Web of Science ID A1994BB16D00020
Ovulation and subsequent luteal tissue formation are preceded by midcycle surges of both LH and FSH. Although LH has been widely known as the luteinizing hormone, a potential role for FSH in the luteinization process is possible. Our earlier studies using recombinant FSH (rcFSH) without LH contamination have shown that treatment with a surge dose of rcFSH induces ovulation of mature follicles in hypophysectomized rats. The present studies examined further whether FSH alone is sufficient to induce normal corpus luteum formation. Immature hypophysectomized rats were implanted with an estrogen pellet (10 mg diethylstilbestrol). Two days later, a minipump releasing 4 IU rcFSH/day was placed to induce follicular growth. Forty-eight hours after FSH treatment, both DES pellet and FSH minipump were removed, and rats were injected with a single sc dose of 40 IU rcFSH, 5 micrograms hCG, or saline. For some animals, oviducts were excised the following day to determine the number of ovulated oocytes. The remaining animals received, 2 days later, sc injections of 125 micrograms ovine PRL twice daily for 3 days to maintain luteal function. All rats that received a surge dose of rcFSH or hCG ovulated similar numbers of oocytes, whereas none of the control animals did. Ovaries and blood samples were obtained 5 days after the gonadotropin surge. rcFSH and hCG significantly increased ovarian weight to 73.9 +/- 4.8 and 94.7 +/- 5.6 mg, respectively, compared to 10.0 +/- 0.5 mg in controls. Serum progesterone levels were increased by 192- and 102-fold in rcFSH- and hCG-treated animals, respectively, compared with those in the saline-treated rats. rcFSH and hCG also induced a marked elevation of ovarian [125I]hCG binding (4.2 +/- 0.2 and 3.7 +/- 0.1 ng/mg ovary, respectively), whereas ovaries from control animals exhibited low binding (0.6 +/- 0.1 ng/mg ovary). These gonadotropin-induced increases in [125I]hCG binding were associated with similar elevations in the levels of three LH receptor transcripts of 2.5, 4.2, and 7.0 kilobases. Also, levels of the ovarian cholesterol side-chain cleavage enzyme (CYP 11A) mRNA (2 kilobases) were low in control animals, but increased 20.5- and 14.3-fold after surge doses of rcFSH and hCG, respectively. Accompanied by biochemical signs of luteinization, morphological features typical of luteinized ovaries were found in both rcFSH and hCG groups, showing the formation of large polyhedral lutein cells and small spindle-shaped lutein cells.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1993MK15100068
View details for PubMedID 8243314
Because of the microheterogeneities of gonadotropins, measurement of immunoreactivity of these glycoproteins does not necessarily reflect changes in their bioactivity. In addition, LH bioactivities in human samples analyzed by a rodent LH bioassay have been discordant with findings based on human granulosa-luteal cells. We have isolated a human LH/choriogonadotropin (CG) receptor cDNA and expressed the recombinant protein. Using 293 cells permanently transfected with the human LH receptor cDNA and a luciferase reporter gene driven by a cAMP-dependent promoter, we have developed a luminescence LH/CG bioassay. After cells were treated with human LH or CG for 20 h, luciferase activity was measured through use of a luminometer. Luciferase activity in the cells was increased in a dose-dependent manner. In contrast, treatment with FSH, thyroid-stimulating hormone, prolactin, growth hormone, adrenocorticotropin, insulin, prostaglandins, and several neurotransmitters had no effect. Because treatment with basic fibroblast growth factor (bFGF) caused significant increases in basal luciferase activity, a fixed amount of bFGF was included in all reactions. Incubation with 0.1 to 30 microliters serum from women during different physiological states stimulated the luciferase activity in parallel with the hCG standard curve. In 4 conception cycles, bioactive LH/hCG levels began to increase 2 wk after the midcycle LH surge, followed by a logarithmic increase from 22 days on. Due to the lack of a homologous RIA for measuring CG levels in monkeys, we analyzed serum bioactive monkey CG (mCG) in macaque during early pregnancy. Bioactive mCG was detected about 12 days after the midcycle LH surge and fertile mating and persisted until Days 21-23, followed by a decline.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1993MH46900020
View details for PubMedID 8286613
Apoptotic cell death has recently been suggested to be the underlying mechanism of ovarian follicle atresia. To study the regulation of follicle cell apoptosis by sex steroids, we have analyzed ovarian DNA fragmentation, the hallmark of apoptosis, in rats treated with estrogens and androgens. Immature rats were hypophysectomized and implanted with diethylstilbestrol (DES) capsules. Two days later, DES implants were removed in some animals, followed by treatment with estrogens with or without androgens. The extent of ovarian apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. After DES withdrawal, ovarian weight decreased and DNA fragmentation increased in a time-dependent manner. In granulosa cells, an increase in apoptotic DNA fragmentation was seen 12 h after withdrawal of DES implants, followed by a 25-fold increase at 48 h. In situ analysis of DNA fragmentation on histological sections of ovaries, using a nonisotopic labeling of DNA by digoxigenin-dideoxy-UTP, also demonstrated that apoptosis induced by DES withdrawal is confined to the granulosa cells in early antral and preantral follicles. No increase in DNA breakdown was detected in thecal cells and interstitial tissues or granulosa cells of primordial and primary follicles. In contrast, replacement with DES (0.5 mg twice daily) or estradiol benzoate (3 mg daily) completely prevented the observed ovarian weight loss and increases in granulosa cell apoptosis. Treatment with estradiol benzoate (0.003-3 mg/day) dose dependently suppressed the apoptosis seen 2 days after removal of DES implants. Furthermore, the antiatretogenic effect of estrogen was blocked by treatment with testosterone (0.5 mg twice daily), which increased ovarian apoptotic DNA fragmentation and decreased ovarian weight in DES-treated animals in a time-dependent manner. Also, in situ examination showed that androgen treatment increased apoptosis in the granulosa cells in a subpopulation of early antral and preantral follicles. The specificity of testosterone action was further demonstrated by the lack of effect of progesterone and cortisol on ovarian apoptosis. These data suggest that sex steroids play an important role in the regulation of ovarian apoptotic cell death, with estrogens preventing apoptosis and androgens antagonizing the effect of estrogens. These data provide the basis for future studies on the role of sex steroid hormones in follicular atresia and the regulation of endonuclease activity by steroid hormones.
View details for Web of Science ID A1993ME08000038
View details for PubMedID 8404672
Proteolytic activity generated by the plasminogen-activator system (PA system) is associated with many biological processes. However, it is not known how the proteolytic activity is regulated in vivo in order to obtain directed proteolysis while, at the same time, protecting unrestrained tissue destruction. Using gonadotropin-induced ovulation as a model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen-activator-inhibitor type 1 (PAI-1), are regulated temporally and spatially by gonadotropins, leading to the initiation and termination of a well-directed proteolytic process. In-situ hybridization and in-situ zymography were used to analyze the expression of tPA and PAI-1 mRNA and PA-activity in specific ovarian cell types. Both tPA and PAI-1 were found to be regulated and to have a distinct expression pattern in different ovarian compartments. tPA was expressed in both granulosa and thecal-interstitial cells; the highest levels of tPA mRNA were found in the granulosa cells of preovulatory follicles, just prior to ovulation. Consistent with a role for luteinizing hormone/chorionic gonadotropin (LH/CG) in triggering ovulation, the cells and follicles that actively expressed tPA also contained high levels of LH-receptor mRNA while cumulus cells that contain undetectable amounts of tPA mRNA were devoid of LH-receptor expression. The highest levels of PAI-1 mRNA were found about 6 h before ovulation and mainly in the thecal-interstitial cells and ovarian stroma tissue which encapsulate the follicle. Preovulatory follicles, protruding onto the surface of the ovary with less surrounding stroma tissue, expressed less PAI-1 compared to small non-ovulatory follicles embedded in inner part of the ovary. In-situ zymography also revealed that the PA activity was colocalized to the surface of the ovary just prior to ovulation. Our studies suggest that a proteolytic activity provided by tPA and modulated by PAI-1 is responsible for a controlled and directed proteolysis leading to rupture of selected follicles during ovulation.
View details for Web of Science ID A1993LE43600019
View details for PubMedID 8508786
Although the requirement for pituitary gonadotropins during testicular cell differentiation is well documented, the possible role of FSH and LH in regulating testicular cell survival has not been studied. Using a quantitative autoradiographic method for the detection of internucleosomal DNA fragmentation, a hallmark feature of apoptosis, the hormonal control of apoptotic cell death was studied in testicular cells collected from immature rats after hypophysectomy. After surgery, animals were treated with daily injections of 20 IU long-acting FSH agonist (FSH-CTP) or 50 IU human CG (hCG) for 2 days. Hypophysectomy decreased testis weight by 25%, but treatment with FSH-CTP or hCG prevented the effect of hypophysectomy. Testes of intact animals contained predominantly high-mol wt DNA, whereas hypophysectomy increased DNA cleavage into low-mol wt (< 15 kilobases) ladders characteristics of apoptosis. In contrast, treatment with FSH-CTP or hCG inhibited hypophysectomy-induced apoptotic DNA cleavage by 84% and 51%, respectively. Hypophysectomy-induced DNA fragmentation was found in both interstitial cells and seminiferous tubules. Similar to whole testis, treatment with FSH-CTP suppressed hypophysectomy-induced apoptosis by over 90% in seminiferous tubules and interstitial cells. In contrast, hCG treatment was less effective in preventing hypophysectomy-induced DNA cleavage (46% suppression in tubules and 77% suppression in interstitial cells). Furthermore, testosterone replacement also suppressed hypophysectomy-induced DNA fragmentation by 75% in the whole testis tissue, 64% in tubules, and 55% in interstitial cells. To further study the role of gonadotropins, intact animals were treated with a potent GnRH antagonist (Azaline B, 10 microgram/day) to decrease serum gonadotropin levels.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1993LD85600002
View details for PubMedID 8316250
A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or "programmed cell death." This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [alpha 32P]-dideoxynucleotide to the 3'-end of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells.
View details for Web of Science ID A1993KP60400009
View details for PubMedID 8436601
View details for Web of Science ID A1993KP97400305
In the rat, the antagonistic properties of deglycosylated (dg) gonadotropins in vitro are characterized by high affinity receptor binding but impaired ability to stimulate cAMP accumulation. In human, the functional role of N-linked sugars in human CG (hCG) action is unclear because of the unavailability of totally deglycosylated hCG and because of the difficulty involved in obtaining human gonadal tissues. We have recently prepared completely deglycosylated hCG using site-directed mutagenesis and expressed functional human LH (hLH) receptors using cloned complementary DNA. Since hLH receptor shows distinct ligand specificity from that of rat LH receptor, we examined binding kinetics and signal transduction of recombinant dg-hCG using recombinant hLH receptors. In embryonic human kidney cells (293) transfected with hLH receptor complementary DNA, 125I-hCG binding to its receptor was studied in the presence of varying amounts of unlabeled dg-hCG or wild type (WT)-hCG. Lineweaver-Burk analysis of the binding kinetics showed that the displacement of 125I-hCG by dg-hCG was noncompetitive whereas that seen for WT-hCG was competitive. The noncompetitive nature of dg-hCG binding was further confirmed using rat LH receptors present in testis membrane preparations. After preincubation of LH receptor-expressing 293 cells with WT-hCG, inclusion of 125I-hCG competitively displaced WT-hCG. In contrast, preincubation with dg-hCG prevented subsequent 125I-hCG binding to human LH receptor for at least 46 h. WT-hCG caused a dose-dependent increase in cAMP accumulation in the 293 cells with an ED50 of 10 ng/ml. However, dg-hCG was ineffective in inducing cAMP production with a maximal effect of only 12% of that stimulated by WT-hCG. In the presence of increasing doses of dg-hCG, stimulation of cAMP by WT-hCG was antagonized in a dose-dependent manner. In contrast, forskolin stimulation of cAMP was not antagonized by dg-hCG, indicating receptor-mediation of dg-hCG action. Similar to binding studies, preincubation with dg-hCG also dose-dependently blocked the subsequent stimulatory effect of WT-hCG on cAMP production. Thus, the noncompetitive binding of dg-hCG to hLH receptors and its antagonism of hCG stimulation of cAMP accumulation suggest that dg-hCG is an irreversible receptor blocker with unique antagonistic properties.
View details for Web of Science ID A1993KJ81900042
View details for PubMedID 8381073
View details for Web of Science ID A1993BC44T00027
View details for Web of Science ID A1993BZ01H00024
The induction of granulosa cell differentiation and follicle maturation is dependent upon the stimulatory actions of FSH. Our recent studies used recombinant DNA technology to fuse the carboxyl-terminal peptide (CTP) of hCG beta-subunit to the carboxyl-terminus of the FSH beta-subunit. The resulting FSH analog has identical in vitro receptor-binding and biological activities as wild-type FSH (WT-FSH), but an increased circulating half-life. The present studies examined further the ability of FSH with one (FSH-CTP1) or two (FSH-CTP2) appended CTPs to promote granulosa cell differentiation and follicle ovulatory potential. WT-FSH, FSH-CTP1, and FSH-CTP2 were produced from Chinese hamster ovary cells transfected with the common alpha-subunit and respective beta-subunit. Hormone concentrations were quantitated by RIA, and relative levels confirmed by radioligand receptor assay. Both FSH-CTP1 and FSH-CTP2 retained full FSH receptor-binding activity, but did not bind LH receptors. To compare in vivo bioactivity, immature estrogen-primed female rats received ip injections of FSH or the agonists at 0 and 24 h. At 48 h, substantial stimulation (up to 2.5-fold) of ovarian weight was induced by 1.0 and 3.0 IU/day FSH-CTP1 or FSH-CTP2, whereas a higher dose (10 IU/day) of WT-FSH was required for an 1.8-fold stimulation. Although the in vivo potencies of FSH-CTP1 and FSH-CTP2 were similar, FSH-CTPs were about 10-fold more potent than WT-FSH in inducing granulosa cell aromatase activity and LH receptors. We further reduced the frequency of hormone administration. Increasing doses (1-10 IU) of a single ip injection of FSH-CTP1 resulted in dose-dependent increases in granulosa cell aromatase activity and LH receptor content 48 h later. Although a single injection (10 IU) of WT-FSH had no effect, the same total dose of WT-FSH administered as four 2.5-IU injections 12 h apart was effective. To test the ovulatory potential of ovarian follicles, rats received a single injection of FSH-CTP1, followed 52 h later by 5 IU hCG to induce ovulation. Although hCG did not induce ovulation in females receiving a single dose (10 IU) of WT-FSH, 20 +/- 2 and 43 +/- 5 ovulated ova/rat were found in animals primed with 3 and 10 IU FSH-CTP1, respectively. Because twice daily injections of WT-FSH (2.5 IU/injection) also increased the ovulatory potential of the ovary, the enhanced effectiveness of FSH-CTP1 appears to be related to its increased circulating half-life.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1992KB06800004
View details for PubMedID 1446593
The inhibins are alpha:beta heterodimeric growth factors that are members of the transforming growth factor-beta family. To understand the physiological roles of the inhibins in mammalian development and reproduction, a targeted deletion of the alpha-inhibin gene was generated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the null allele (inhibin-deficient) initially develop normally but every mouse ultimately develops mixed or incompletely differentiated gonadal stromal tumours either unilaterally or bilaterally. Inhibin is thus a critical negative regulator of gonadal stromal cell proliferation and the first secreted protein identified to have tumour-suppressor activity.
View details for Web of Science ID A1992JZ63000041
View details for PubMedID 1448148
Recent biochemical studies have suggested that apoptotic cell death is the molecular mechanism underlying the degeneration of ovarian follicles during atresia. Using a sensitive autoradiographic method for the detection of DNA fragmentation, we studied apoptosis in ovarian granulosa cells or intact follicles placed in serum-free culture as model systems to elucidate the hormonal regulation of atresia. Immature rats (25 days old) were primed for 2 days with 10 IU equine CG to induce a homogeneous population of mature preovulatory follicles. Granulosa cells isolated from these follicles contained predominantly intact high mol wt DNA. However, a time-dependent, spontaneous onset of internucleosomal DNA fragmentation characteristic of apoptotic cell death occurred in granulosa cells during culture. Treatment of granulosa cells with epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), or basic fibroblast growth factor (bFGF) inhibited the spontaneous onset of apoptotic DNA cleavage found during culture by 40-60%. In contrast, insulin-like growth factor I, insulin, TGF beta and tumor necrosis factor-alpha were ineffective. Likewise, activation of the protein kinase A or C pathways with forskolin or phorbol 12-myristate 13-acetate, respectively, did not prevent the onset of DNA fragmentation, although inclusion of a tyrosine kinase inhibitor (genistein) completely blocked the ability of EGF, TGF alpha, and bFGF to suppress apoptosis in granulosa cells. Similar to cultured granulosa cells, a spontaneous onset of apoptosis was also observed to occur in isolated preovulatory follicles during culture. Furthermore, treatment of follicles with EGF or bFGF inhibited the spontaneous initiation of apoptosis, and the suppressive effects of these growth factors were also attenuated by co-treatment with genistein.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992KB01500021
View details for PubMedID 1480180
Although atresia of ovarian follicles is of critical importance during preovulatory follicle selection as well as during normal and premature menopause, the mechanisms underlying atresia remain poorly understood. To study molecular events associated with atresia, we evaluated changes in mRNA levels for cytochrome P450 aromatase, FSH receptor, LH receptor, and a structural protein, beta-actin, during atresia in small (3-mm diameter) and large (6-mm diameter) porcine follicles. In addition, internucleosomal fragmentation of DNA characteristic of apoptosis ("programmed cell death") was assessed in individual healthy and atretic follicles using a sensitive autoradiographic method. Follicles were classified as morphologically healthy or atretic based on the absence or presence of follicular haemorrhagia and the degree of follicular clarity. Morphological signs of atresia in individual follicles were correlated with the occurrence of internucleosomal DNA fragmentation in granulosa cells as well as in thecal cells during advanced stages of atresia. The presence of apoptosis in atretic follicles was also associated with significant decreases in follicular fluid estrogen concentrations compared to those in healthy follicles of the same size. The decline in estrogen synthesis in degenerating follicles was further correlated with decreased levels of a predominant 2.6-kilobase aromatase mRNA. Moreover, substantial declines in both FSH receptor and LH receptor mRNAs were found in atretic follicles, consistent with previous reports of their decreased responsiveness to gonadotropins. The observed decreases in mRNAs for aromatase and gonadotropin receptors could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of intact 18S and 28S ribosomal RNA as well as constitutive expression of beta-actin mRNA in atretic follicles. These data indicate that apoptotic cell death is initiated in both granulosa and thecal cells of porcine follicles during atresia. Associated with internucleosomal DNA fragmentation, decreased transcription of specific ovarian genes or destabilization of their transcripts leads to selective decreases in aromatase and gonadotropin receptor mRNAs. The atresia of ovarian follicles provides an interesting model to further study the molecular events associated with DNA fragmentation and selective mRNA down-regulation during apoptosis.
View details for Web of Science ID A1992JT90400016
View details for PubMedID 1396312
The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.
View details for Web of Science ID A1992JG58100039
View details for PubMedID 1322283
Inhibin suppresses the pituitary secretion of FSH but not LH. The two forms of inhibin are composed of a common alpha subunit linked to either a beta A or a beta B subunit. The mouse inhibin alpha gene was isolated and shown to have two exons spanning a 1.7 Kb intron. The proximal 5' flanking region has neither TATA and CAAT boxes nor GC-rich area. Using the 5' flanking region of mouse inhibin alpha gene linked to luciferase gene, transfection of rat granulosa cells indicated that the first 165 bp of the promoter region is required for basal expression. The mouse inhibin alpha genomic clone should be useful for analysis of hormonal control of inhibin alpha transcription and the generation of mice with targeted deletion of this gene.
View details for Web of Science ID A1992JE59900040
View details for PubMedID 1632772
The anterior pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), act upon the ovary and testis via occupancy of specific cell membrane receptors, resulting in increased cAMP production, steroidogenesis, and expression of differentiation-related genes. Recent cloning of the cDNAs for LH and FSH receptors allows the analysis of mRNA levels for these receptors in gonadal tissues. This review summarizes progress in elucidating the molecular basis of LH and FSH receptor gene regulation in the ovary and testis during different physiologic states.
View details for Web of Science ID A1992JB09200003
View details for PubMedID 18407096
On the basis of earlier observations of changing testicular LH receptor levels during postnatal development of the rat, we analyzed the levels of LH receptor mRNA transcripts in testes of rats at ages 5-70 days. Extracted testis RNA, prepared for Northern blotting, was hybridized with a specific LH receptor cRNA probe derived from subcloned cDNA corresponding to the extracellular domain of the receptor. Six LH receptor mRNA transcripts with molecular sizes of 7.8, 7.0, 4.2, 2.5, 1.8, and 1.2 kb were identified. Of these, the 1.2- and the 1.8-kb mRNA transcripts presumably code for truncated forms of LH receptor. At 5 days, only the 1.8- and the 4.2-kb mRNA transcripts were observed. Additional 7.0- and 1.2-kb transcripts appeared at 10 and 15 days, respectively. From the age of 25 days through adulthood, all six mRNA transcripts were observed. Densitometric analyses revealed that the amounts of the 7.0- and 1.8-kb mRNA transcripts correlated well with LH receptor levels, while the 4.2-kb transcript showed high levels earlier in life with poor correlation to LH receptor number. Because the 1.8 kb receptor transcript lacked transmembrane domains, the present results suggest the 7.0-kb LH receptor transcript as the likely candidate to encode the functional receptor. These data provide the basis for future analyses of the molecular regulation of LH receptor expression.
View details for Web of Science ID A1992HV72700005
View details for PubMedID 1391300
Follitropin (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of anovulatory women. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, we constructed chimeric genes containing the sequence encoding the C-terminal peptide of the chorionic gonadotropin beta subunit (CG beta) fused to the translated sequence of the human FSH beta subunit (FSH beta). This region of CG beta is important for maintaining the prolonged plasma half-life of human CG dimer. The presence of the C-terminal peptide sequence did not significantly affect assembly of FSH beta with the alpha subunit or secretion of the dimer. In vitro receptor binding and steroidogenic activity of dimer bearing the FSH beta-C-terminal peptide chimera were the same as wild-type FSH. However, both the in vivo potency and half-life in circulation of the dimer bearing either one or two C-terminal peptide units were enhanced. Dimers containing FSH beta-CG beta chimeras could serve as potent FSH agonists for clinical use, and the present strategy may have wide applications for enhancing the in vivo half-life of diverse proteins.
View details for Web of Science ID A1992HU97700018
View details for PubMedID 1374895
View details for PubMedCentralID PMC49070
The maturation of ovarian granulosa cells is dependent upon the pituitary gonadotropin FSH, the actions of which are mediated via specific plasma membrane receptors. To study the regulation of ovarian FSH receptor expression at the mRNA level, we used a specific cRNA probe to evaluate changes in FSH receptor transcripts in cultured granulosa cells. Granulosa cells obtained from immature estrogen-treated rats contained two predominant FSH receptor mRNA transcripts (7.0 and 2.5 kilobases), the levels of which declined in a time-related manner during a 2-day culture period. However, inclusion of FSH (30 ng/ml) in the culture medium prevented the decline in FSH receptor mRNA levels. Compared to controls, treatment of granulosa cells for 48 h with FSH (1-100 ng/ml) increased FSH receptor mRNA levels in a dose-dependent manner (ED50, 4.5 ng/ml), with a maximal 5.9 +/- 0.7-fold increase observed in response to 30 ng/ml FSH. The stimulatory actions of FSH were mimicked by the adenyl cyclase activator forskolin (0.1-30 microM), suggesting the involvement of cAMP in FSH receptor gene transcription and/or mRNA stability. Incubation of granulosa cells for 48 h with epidermal growth factor (EGF; 0.3-10 ng/ml), basic fibroblast growth factor (bFGF; 1-30 ng/ml), or insulin-like growth factor-I (IGF-I; 1-30 ng/ml) did not affect basal FSH receptor mRNA levels, whereas the highest doses of EGF and bFGF, but not IGF-I, completely suppressed the stimulatory effects of FSH (30 ng/ml) on its own receptor mRNA levels. Similarly, GnRH (10-1000 nM) attenuated the actions of FSH on its receptor mRNA levels in a dose-dependent manner (ID50, 8 nM). The inhibitory effects of GnRH (100 nM) were reversed by cotreatment with a GnRH antagonist ([Ac-D-Phe1,D-pCl-Phe2,D-Trp3,6]GnRH; 100 nM), indicating that the actions of GnRH are mediated via specific GnRH receptors. These data indicate that treatment of granulosa cells with FSH increases the levels of two FSH receptor mRNA transcripts. However, this positive feedback system, which may lead to an amplification of FSH action, is tightly regulated by the inhibitory actions of EGF, bFGF, and GnRH. Thus, the use of cultured rat granulosa cells provides a model system to analyze the hormonal regulation of FSH receptor gene expression in the ovary.
View details for Web of Science ID A1992HG16100029
View details for PubMedID 1311235
The actions of gonadotropins on ovarian differentiation are associated with dynamic changes in gonadotropin receptor content, presumably due to modulation of receptor gene expression. The present studies used a reverse transcription-polymerase chain reaction to obtain a rat FSH receptor cDNA fragment, followed by synthesis of a labeled cRNA probe to examine the regulation of FSH receptor mRNA levels during follicular maturation, ovulation, and luteinization. Northern blot analysis of ovarian RNA with the FSH receptor probe revealed two predominant hybridization signals of 7.0 and 2.5 kilobases (kb) as well as minor signals of 4.2 and 1.8 kb. Treatment of immature rats with PMSG (10 IU) to induce follicular development resulted in increased FSH receptor mRNA levels 24 h after treatment, with a further increase at 52 h, coincident with increased [125I]FSH binding. Subsequent treatment with an ovulatory dose of hCG decreased FSH binding and receptor mRNA levels by 6 h, with a maximal inhibition at 24 h after hCG. In luteinized ovaries obtained 3 and 5 days after hCG treatment, the 7.0-kb FSH receptor mRNA increased again, but no concomitant elevation of [125I]FSH binding was detected. We recently demonstrated that FSH treatment alone is capable of inducing follicular growth and ovulation, thus providing a unique model to evaluate the effects of FSH on regulation of its receptor gene. Immature hypophysectomized estrogen-treated rats were implanted with an osmotic minipump delivering recombinant human FSH (rcFSH; 4 IU/day) to stimulate follicle growth, followed 52 h later with a single injection (20 IU) of rcFSH to induce ovulation. Stimulation of follicular growth with rcFSH increased both FSH receptor binding and mRNA levels. In contrast, the ovulatory dose of rcFSH decreased FSH binding and receptor message levels within 12 h. Thus, gonadotropin regulation of ovarian FSH receptor content during follicular growth, ovulation, and luteinization is associated with similar changes in FSH receptor message levels. Also, studies using rcFSH demonstrate that both up- and down-regulation of FSH receptor gene expression can be induced by the homologous hormone at different stages of follicle development.
View details for Web of Science ID A1992HG16100028
View details for PubMedID 1537292
View details for Web of Science ID A1992HD85500008
View details for Web of Science ID A1992JD06600023
View details for Web of Science ID A1992BY12C00018
The action of LH is mediated through specific plasma membrane receptors that are both up- and down-regulated in the ovary during the reproductive cycle. Using immature rats treated with PMSG and hCG as a model system, we have studied the regulation and distribution of LH receptor mRNA in different cell types during follicle development, ovulation, and luteinization by Northern blot and in situ hybridization. In untreated rats, LH receptor mRNA was below the detection level in granulosa cells, cumulus cells, and oocytes, while low levels of LH receptor mRNA were found in the thecal cells. After stimulation with PMSG, expression of LH receptor mRNA was enhanced in the thecal-interstitial cells, while a more dramatic increase in receptor mRNA abundance took place in granulosa cells of large tertiary follicles. In these follicles, the abundance of LH receptor mRNA varied among different subpopulations of granulosa cells, with mural granulosa cells close to the basement membrane exhibiting higher levels than granulosa cells located closer to the antrum, and cumulus cells and the oocyte lacking detectable hybridization signal. The uneven expression of LH receptor mRNA endows different ovarian cells with varying hormonal responsiveness. After an ovulatory dose of hCG, LH receptor mRNA levels were dramatically decreased, particularly in the granulosa cells of preovulatory follicles, to reach the lowest levels just before ovulation. During the transformation of ovulated follicles into corpora lutea, the expression of LH receptor message was again increased. Our results reveal that the previously documented regulation of the LH receptor-binding activity during ovarian development correlates with expression of the LH receptor transcripts, suggesting that the LH receptor gene is regulated in a complex manner during the periovulatory period to achieve cell-specific expression together with gonadotropin induction and suppression of receptor gene activity.
View details for Web of Science ID A1991GT67000051
View details for PubMedID 1954899
View details for PubMedID 19912830
In the ovary, greater than 99% of the follicles present at birth are destined to degenerate during life. In humans, less than 400 of the more than 400,000 follicles found at puberty will eventually ovulate whereas the overwhelming majority of follicles undergo atresia. Although follicular atresia plays a critical role during the recruitment of follicles for ovulation, the exact mechanism of this process is unknown. In chicken and porcine ovaries, atretic follicles can be morphologically distinguished from their healthy counterparts of the same size. Adapting a sensitive 3'-end labeling method for DNA analysis, we identified internucleosomal cleavage of cellular DNA in atretic (but not normal) follicles of both animal species, resembling that found during programmed cell death in embryogenesis, autoimmune T-cell removal and prostate regression. The present findings provide a basis for elucidating the hormonal signals involved in the initiation of follicular atresia during follicle recruitment, reproductive aging and premature ovarian failure.
View details for Web of Science ID A1991GM34900077
View details for PubMedID 1718732
Although earlier reports suggest a stimulatory effect of FSH on Leydig cell function, controversy exists due to unavailability of FSH preparations free of contaminating LH. Recent availability of recombinant human FSH preparations made it possible to reinvestigate this question. Immature male rats were hypophysectomized (21-22 days old at surgery) and implanted with osmotic minipumps releasing 8 IU recombinant FSH or 18 IU purified human pituitary FSH (hpFSH)/day, whereas control animals received vehicle alone. After 7 days of treatment, testicular weight increased in the recombinant FSH and hpFSH-treated animals to values 2.3- and 2.5-fold those of controls, respectively. Analyses of the steroidogenic capacity of Leydig cells in testes of rats treated with recombinant FSH or hpFSH also revealed 2.9- and 3.8-fold androgen production in vitro compared to controls. In these rats recombinant FSH and hpFSH increased the LH receptor number in testicular homogenate by 50% and 70%, respectively. The increase in LH receptor number was associated with increases in the LH receptor mRNA levels. In hypophysectomized control rats, small seminiferous tubules contained spermatogonia and zygotene/early pachytene spermatocytes. In contrast, treatment with either FSH preparation enhanced the progression of meiosis, as evidenced by large number of pachytene spermatocytes and appearance of round spermatids. The present results show that LH-free recombinant FSH, like purified pituitary FSH, is capable of increasing the LH receptor content and steroidogenic responsiveness of Leydig cells through paracrine mechanisms together with a stimulatory effect on spermatogenesis. These observations suggest that prepubertal elevation of FSH secretion may be important for increasing Leydig cell steroidogenic capacity and spermatogenic progression.
View details for Web of Science ID A1991GH33600035
View details for PubMedID 1915076
Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two post-translationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine alpha and FSH beta subunits and a modified rat beta-casein gene-based expression system. Lines of bigenic mice expressing both subunits have been generated either by coinjection of the subunit transgenes or by mating mice that acquired and expressed transgenes encoding an individual subunit. Up to 60 international units (15 micrograms) of biologically active FSH per ml was detected in the milk of the bigenic mice. These lines provide a model system for studying the post-transcriptional mechanisms that effect the expression and secretion of this heterodimeric hormone.
View details for Web of Science ID A1991GH80500010
View details for PubMedID 1924292
View details for PubMedCentralID PMC52501
Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.
View details for Web of Science ID A1991FU00500003
View details for PubMedID 1922095
The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation.
View details for Web of Science ID A1991FJ39600033
View details for PubMedID 1902167
Down-regulation of plasma membrane receptors by homologous hormones has been found in diverse cell types. In testicular Leydig and ovarian luteal cells, treatment with LH/hCG decreases LH receptor content. Although suppression of LH-binding sites may result from ligand-induced receptor internalization, sequestration, and/or phosphorylation, the gonadotropins may also regulate receptor mRNA levels. We examined the regulation of testis LH receptor mRNAs in adult rats that received 10 or 200 IU hCG, using cRNA probes derived from the 5' extracellular domain (EC) or the 3' transmembrane domain (TM) of the rat receptor cDNA. Probe EC hybridized to predominant signals of 7 and 1.8 kilobases (kb) and weaker signals of 4.2 and 2.5 kb. However, probe TM hybridized to the three larger forms of the LH receptor mRNA, but not to the 1.8-kb species, suggesting that the latter form lacks the transmembrane domain. After 6 and 12 h of treatment with 200 or 10 IU hCG, respectively, hybridization to the larger mRNA species decreased by more than 60%, preceding decreases in testicular [125I]hCG binding. These transcripts were further inhibited (greater than 93%) between 24-72 h after hCG treatment and returned to 40% and 100% of control levels by days 6 and 9, respectively. In contrast, the truncated 1.8-kb LH receptor transcript was not affected by hCG treatment, indicating a differential suppressive effect of the ligand on its receptor mRNA levels. In the ovary, hybridization to probe EC revealed four transcripts with similar sizes as those found in the testes, with a predominant 7-kb species.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1991FE67400010
View details for PubMedID 1890990
Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common alpha- and FSH beta-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.
View details for Web of Science ID A1990EL10800053
View details for PubMedID 2123446
Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic fibroblast growth factor (bFGF), an angiogenic factor found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the 22S tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in growing follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.
View details for Web of Science ID A1990EF51500043
View details for PubMedID 2171912
Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.
View details for Web of Science ID A1990DQ68500043
View details for PubMedID 2115434
Recent studies have shown that Chinese hamster ovary (CHO) cells transfected with the FSH subunit genes secrete bioactive FSH. Here, we determined the in vitro and in vivo bioactivity of recombinant FSH produced by CHO mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase-I (NAGT-), resulting in glycoproteins with asparagine-linked (GlcNAc)2(Mannose)5 oligosaccharides, or mutant cells defective in sialic acid transport into the Golgi (ST-). In the latter, glycoproteins are secreted lacking terminal sialic acids. Determination of in vitro bioactivity, using the granulosa cell aromatase bioassay, indicated that both FSH variants are as active as FSH secreted by the wild type (WT) cells and purified pituitary FSH. Also, these normal and variant forms of FSH are equipotent in a radioligand receptor assay using rat testis membranes. However, the variant FSH molecules are more basic than the WT FSH as determined using a chromatofocusing column (pI: wild type 3.6-5.0, NAGT- greater than 7.0, ST- approximately 6.0 and greater than 7.0). Injection of immature estrogen-treated rats with WT FSH induced high aromatase activity in their granulosa cells whereas treatment with either one of the FSH variants was ineffective; the lack of in vivo activity of the FSH variants was correlated with rapid clearance of these molecules in serum. Thus, recombinant human FSH produced by cells deficient in NAGT-I or defective in sialic acid transport retains normal receptor binding and in vitro bioactivity, but exhibits minimal in vivo activity and a shortened half-life when compared to WT FSH, indicating the important role of terminal sugars for FSH action in vivo.
View details for Web of Science ID A1990DM02300015
View details for PubMedID 2141816
Tissue-type plasminogen activator (tPA) is secreted by rat granulosa cells in response to treatment with activators of protein kinase A (follitropin, FSH), protein kinase C (gonadotropin-releasing hormone, GnRH) and tyrosine kinase (epidermal growth factor, EGF). Because steroid hormones have been shown to enhance the gonadotropin stimulation of ovarian differentiation, we investigated the effects of steroid hormones, alone or together with various kinase activators, on tPA activities and mRNA levels in cultured rat granulosa cells. Treatment of cells with dexamethasone (DEX; a glucocorticoid agonist) or R1881 (an androgen agonist) caused an increase in tPA secretion and mRNA levels. In addition, the stimulation of tPA activity and mRNA levels by FSH (50 ng/ml) was synergistically enhanced by cotreatment with DEX or R1881 in a time-dependent manner with 2.8- and 1.6-fold increase at 9 h after incubation as compared to cells treated with FSH alone. In contrast, treatment with diethylstilbestrol had no effect on tPA levels. Furthermore, tPA activity and mRNA levels induced by GnRH and EGF were also increased by cotreatment with DEX or R1881 as compared with cells treated with GnRH or EGF alone. Likewise, the stimulation of tPA mRNA levels by dibutyryl cAMP, a protein kinase A activator, and phorbol myristate acetate (PMA), a protein kinase C activator, was enhanced by cotreatment with DEX or R1881. These results demonstrate that glucocorticoid and androgen enhance tPA secretion and mRNA levels stimulated by FSH, GnRH and EGF in granulosa cells. The rat granulosa cells provide a useful model for studying the mechanism of regulation of tPA gene expression by steroid hormones.
View details for Web of Science ID A1990CM90700010
View details for PubMedID 2107107
View details for Web of Science ID A1990BT23Y00009
View details for Web of Science ID A1990BS21N00002
hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.
View details for Web of Science ID A1990CG37900051
View details for PubMedID 2293995
Rat ovaries produce a novel ovarian trypsin-like protease that is regulated during follicular development. The protease extracted from the ovaries of immature gonadotropin-treated female rats was unstable to denaturation, but was recoverable after non-denaturing electrophoresis. The activity was inhibited by synthetic serine protease inhibitors but not by aprotinin or soybean trypsin inhibitor, thus distinguishing the enzyme from pancreatic trypsin. Treatment with pregnant mare's serum gonadotropin (PMSG) significantly increased the levels of enzyme in the ovarian granulosa cells (Control, 0.0027 units/10(6) cells; PMSG-treated, 0.0062 units/10(6) cells) which was also secreted by these cells. The novel enzyme described here may be important for matrix remodelling during follicular growth.
View details for Web of Science ID A1989CD84400011
View details for PubMedID 2688643
Some modified forms of follicle-stimulating hormone (FSH) may not cross react with the FSH antibody used in the radioimmunoassay (RIA), but still retain their biological activities. Therefore, estimates of immunoreactive FSH may not correspond with FSH bioactivity. The use of in vitro FSH bioassays may disclose possible roles of these heterogenous forms in vivo under various conditions. This review will focus on the history of FSH bioassays, and the widely used in vivo Steelman and Pohley bioassay, based on ovarian weight increase after injection into female rats, is discussed. Since 1971 different biological activities of FSH in various target tissues have been used for the in vitro estimates of FSH bioactivity. The aromatase bioassays involve the measurement of oestradiol in medium of FSH treated Sertoli cells or granulosa cells in culture. Estimates of FSH bioactivity, using the rat granulosa cell bioassay, in serum and urine or normally cycling women, during gonadotropin-releasing hormone-antagonist treatment in hypergonadotropic hypogonadal women and during different stages of female pubertal development will be discussed in more detail.
View details for Web of Science ID A1989CF63800011
View details for PubMedID 2513451
In view of recent reports on ovarian production and action of transforming growth factors (TGFs) and inhibin-related proteins (inhibin, activin, and follistatin), we have examined the effects of these hormones on the function of preovulatory follicles in vitro. Individual preovulatory follicles were obtained from PMSG-treated rats and incubated with these hormones in the absence or presence of LH. Oocyte maturation and progesterone production were monitored. Treatment with TGF alpha alone, but not with TGF beta or inhibin-related proteins, mimicked the action of LH on oocyte maturation by inducing the resumption of meiosis in follicle-enclosed oocytes (56.6% and 80.6% oocytes resumed meiosis in the presence of 0.5 and 1.0 microgram/ml TGF alpha, respectively). In follicle cultures treated with LH to induce oocyte maturation, cotreatment with inhibin and TGF beta (30-50 ng/ml), but not other related hormones, partially inhibited LH-induced meiosis in follicle-enclosed oocytes (from 82% mature ova in the presence of LH to 51% and 55% mature ova with TGF beta and inhibin, respectively). In contrast to follicle cultures, none of the hormones tested significantly affected the spontaneous maturation of rat oocytes explanted from their follicles and cultured within their cumulus mass for 4 h. Treatment with TGF alpha, but not with TGF beta, inhibin, activin, or follistatin, stimulated progesterone production. The present study demonstrated that TGF alpha, like LH, induces oocyte maturation and progesterone production in preovulatory rat follicles. Furthermore, inhibin and TGF beta suppressed LH-induced resumption of meiosis in follicle-enclosed oocytes. Because these growth factors and inhibin-related proteins are synthesized by follicle cells, they may play important roles in regulating follicular development and activity.
View details for Web of Science ID A1989AT94200014
View details for PubMedID 2791970
Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.
View details for Web of Science ID A1989AC10300018
View details for PubMedID 2544397
Interleukin-1 is an important cytokine produced by activated macrophages. Because macrophages have been localized in the testis and interleukin-1 bioactivity has been observed in the testis, the potential effect of interleukin-1 on gonadotropin-stimulated androgen production was investigated using primary cultures of neonatal rat testis cells. Cells were incubated for 3 days before change of medium and treatment with human chorionic gonadotropin and interleukin-1. After 3 additional days medium testosterone and progesterone levels were determined. Human chorionic gonadotropin treatment (0.30-30 micrograms/l) of cultured cells stimulated testosterone production dose-dependently with a maximum increase greater than 18-fold over control values. Although interleukin-1 treatment alone did not affect testosterone production, the concomitant addition of interleukin-1 beta (0.5-5 X 10(3) U/l) caused a dose-dependent decrease of human chorionic gonadotropin action, with 50% inhibition occurring at 1.4 X 10(3) U/l (0.6 X 10(-11) mol/l; N = 5 experiments). Interleukin-1 beta also inhibited forskolin- and dibutyryl cAMP-stimulated testosterone production. The suppression of human chorionic gonadotropin-induced testosterone production by testis cells was accompanied by increased (greater than 3-fold) progesterone levels. Moreover, the conversion of exogenously added androgen precursors (progesterone and 17 alpha-hydroxyprogesterone) to testosterone by human chorionic gonadotropin-stimulated cells was suppressed by interleukin-1 beta suggesting that the activity of the 17 alpha-hydroxylase enzyme may be decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1989U390000001
View details for PubMedID 2541588
To study the structure-function relationships of follitropin (FSH), we expressed the hormone in a heterologous cell system. A genomic clone bearing a 3.7-kilobase FSH beta insert containing the entire coding sequence was transfected alone or together with the alpha subunit gene into Chinese hamster ovary cells and stable lines expressing either FSH beta or FSH dimer were selected. Pulse-chase experiments revealed that, when transfected alone FSH beta was very slowly secreted similar to lutropin beta and thyrotropin beta but unlike choriogonadotropin beta which is efficiently secreted. However, cotransfection of the FSH beta and alpha subunit genes resulted in "rescue" of the beta subunit and rapid secretion of dimer. These data support the hypothesis that the glycoprotein hormones of pituitary origin have determinants for secretion that differ from those on the placental hormone, choriogonadotropin. Recombinant FSH stimulated steroidogenesis comparable to purified human FSH isolated from pituitaries in an in vitro rat granulosa cell assay and appears more homogeneous by chromatofocusing. Human FSH produced by this cell line provides a source of bioactive FSH for experimental and clinical use.
View details for Web of Science ID A1989T780100008
View details for PubMedID 2494176
The biopotencies of pituitary gonadotropins purified from a marsupial (kangaroo), two avian (ostrich and turkey), a reptile (turtle), an amphibian (bullfrog), and two fish (sturgeon and teleost) species were examined using an in vitro rat granulosa cell bioassay for follicle-stimulating hormone (FSH). Treatment of cultured granulosa cells with increasing concentrations of gonadotropin preparations from these species resulted in dose-dependent increases in estrogen production from negligible amounts to maximal levels of approximately 2-29 ng/culture. The relative biopotencies of these FSH preparations from most potent to least potent were in the order of human greater than ostrich greater than turkey greater than kangaroo greater than turtle greater than sturgeon greater than bullfrog greater than teleost with ED50 values of human 8.7 ng/well; ostrich 10.5 ng/well; turkey 22.5 ng/well; kangaroo 58.2 ng/well; turtle 62.5 ng/well; sturgeon 260 ng/well; bullfrog 750 ng/well; teleost greater than 1000 ng/well. In contrast, luteinizing hormone (LH) preparations were considerably less effective for ostrich, turkey, kangaroo, turtle, and bullfrog, being six-, five-, three-, and twofold less potent than FSH preparations for the same species, demonstrating the specificity of this assay for FSH. An LH preparation from bullfrog was unable to significantly stimulate estrogen production below 500 ng/ml. Thus, the present in vitro bioassay (GAB) using rat granulosa cells provides a sensitive and specific assay for measuring FSH activities of gonadotropins from diverse mammalian and nonmammalian species.
View details for Web of Science ID A1989T372000005
View details for PubMedID 2494099
Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin.
View details for Web of Science ID A1989R647400096
View details for PubMedID 2491854
The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.
View details for Web of Science ID A1989R659400028
View details for PubMedID 2462486
View details for Web of Science ID A1989BQ68R00009
Indirect evidence has suggested a role for plasminogen activator (PA) in ovulation. Our recent studies demonstrated that 1) tissue-type PA (tPA) is the predominant PA produced by preovulatory rat follicles in response to gonadotropins or GnRH; and 2) several inhibitors of the serine proteases, to which PA and plasmin belong, block ovulation. Here, the role of tPA and plasmin in ovulation was examined directly by the use of specific antibodies to tPA and alpha 2-antiplasmin (alpha 2AP). Immature female rats at 25-26 days of age were treated (sc) with 15 IU PMSG to induce multiple preovulatory follicles. Fifty-four hours later, tPA antibodies and alpha 2AP were injected into one of the ovarian bursae to check their ability to block ovulation, which was initiated with an ovulatory dose (4 IU) of hCG. The data are expressed as percent inhibition of ovulation in the treated vs. the untreated ovaries. A significant decrease in the ovulation rate was obtained by administration of 500 micrograms antibodies to tPA (39.6%) or 1-50 micrograms alpha 2AP (36-44%), whereas minimal inhibition (12%) was found at lower doses of anti-tPA (10 micrograms) or alpha 2AP (0.1 micrograms). Furthermore, nonimmune immunoglobulin G (500 micrograms) and heat-inactivated alpha 2AP were not effective. Anti-tPA and alpha 2AP suppressed ovulation only when injected at the time of hCG administration; later injections (4-h delay) were ineffective, suggesting that PA and plasmin are involved in the early follicular responses to the ovulatory stimulus. Histological observation of the ovaries did not reveal any pathological changes associated with the anti-tPA and alpha 2AP treatment. Suppression of ovulation, as evidenced by decreased number of tubal ova, was frequently accompanied with intraovarian release of the eggs into the follicular thecal compartment. Thus, these results provide direct evidence for an essential role of tPA and plasmin in ovulation.
View details for Web of Science ID A1989R659400061
View details for PubMedID 2521207
View details for Web of Science ID A1989BR17L00021
View details for Web of Science ID A1989BR17L00012
View details for Web of Science ID A1989BP94X00005
The effect of fibroblast growth factor (FGF) on LH-stimulated testosterone production was investigated using primary cultures of rat testicular cells. Testicular cells obtained from neonatal rats (8-9 days of age) were maintained in culture for 3 days and then challenged with LH with or without basic FGF. After 3 additional days of culture, the media were collected for steroid RIA. LH treatment of cultured cells stimulated testosterone production in a dose-dependent fashion whereas FGF alone did not affect androgen biosynthesis. In contrast, cotreatment with FGF caused a dose-dependent decrease of LH-stimulated testosterone production, with an IC50 value of 1.1 X 10(-9) M (as calculated from three separate experiments). The inhibitory effect of FGF was evident 24 h after the initiation of treatment and this effect was reversible 1 day after the cessation of FGF treatment. The inhibition of LH-induced testosterone production by FGF (maximal inhibition greater than 90%) was accompanied by a 12-fold increase in progesterone levels, suggesting that the inhibitory effect of FGF was distal to the step of progesterone formation. FGF also inhibited forskolin (10(-5) M)- and (Bu)2cAMP (5 X 10(-4) M)-stimulated testosterone production. Furthermore, FGF inhibited the conversion of exogenously added androgen precursors (progesterone and 17 alpha-hydroxyprogesterone) to testosterone in LH-stimulated cultures indicating that FGF might inhibit 17 alpha-hydroxylase activity. The concept of a direct testicular action of FGF was further supported by the demonstration of high affinity (Kd: 3.9 X 10(-10) M; n = 3 experiments) and low capacity (46,900 sites per cell) FGF receptors in cultured testis cells. The binding of [125I]FGF was inhibited by basic and acidic FGF but not by several other growth factors. In conclusion, we suggest that FGF binds to testicular cells and inhibits LH-stimulated testosterone production by inhibiting, at least partially, 17 alpha-hydroxylase enzyme activities. Because FGF has been purified from testis extracts, this growth factor may have intratesticular paracrine or autocrine functions.
View details for Web of Science ID A1988R218000049
View details for PubMedID 2848690
FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.
View details for Web of Science ID A1988P702200011
View details for PubMedID 3139993
The biosynthesis of inhibin in rat granulosa cells was studied by biosynthetic labeling, immunoblotting, and immunocytochemical techniques. Granulosa cells from immature hypophysectomized estrogen-treated rats were cultured in the presence of [35S]cysteine. Both conditioned media and cell extracts were subjected to immunoprecipitation with an antibody directed against the N-terminal 26 amino acids of the alpha-chain of porcine inhibin (pI alpha 1-26), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Treatment with FSH (100 ng/ml) and delta 4-androstenedione (10(-7) M) increased the secretion of 35S-labeled inhibin immunoreactivity by 2.6-fold over that in control cultures treated with androstenedione alone. The radiolabeled inhibin had mol wt (Mr) values of 45,000 and 30,000. Upon reduction, the 45,000 Mr polypeptide remained (with increased apparent Mr of 49,000), but the 30,000 Mr species disappeared with the concomitant appearance of two bands with 18,000 and 11,000 Mr. Competition studies with pI alpha 1-26 confirmed that these polypeptides were all related to inhibin. Furthermore, immunoblotting with an antibody directed against the porcine inhibin beta-A chain (pI beta A81-113) indicated that the 11,000 Mr peptide was the inhibin beta-A chain. Extracts of cells treated with FSH contained only a high Mr alpha-related species (Mr, 41,000 nonreduced; 49,200 reduced). The inhibin alpha antibody was also used to immunocytochemically stain cultured granulosa cells. Cells that had been treated with FSH or the adenyl cyclase activator forskolin (3 x 10(-5) M), but not untreated cells, exhibited positive staining. These results indicate that granulosa cells synthesize and store inhibin alpha-chain precursor with 49,000 Mr. Although some of the high Mr alpha-form was secreted, the majority of the alpha-subunit was processed to the 18,000 Mr form and dimerized with the 11,000 Mr beta-chain to form the mature inhibin dimer immediately before secretion. The cultured granulosa cells may provide a model for future studies on the hormonal regulation of inhibin alpha- and beta-gene expression as well as subunit dimerization and secretion.
View details for Web of Science ID A1988L852600053
View details for PubMedID 3123204
View details for Web of Science ID A1988BR45V00062
The potential role of transforming growth factor-beta (TGF-beta) as an intragonadal regulator in the testis was investigated by studying the effect of TGF-beta on testosterone (T) production by neonatal rat testis cells in primary cultures. After 3 days of preincubation in serum-free medium, testis cells were treated with hormones for 3 additional days. Human chorionic gonadotropin (hCG) treatment (0.3-30 ng/ml) of testis cells elicited a dose-dependent increase of T levels with maximum values greater than 9-fold over baseline. Although TGF-beta alone did not affect T levels, a dose-dependent inhibition of hCG-stimulated T production was observed when cells were cotreated with TGF-beta. Maximal inhibition was greater than 85%, and the IC50 value was 5 ng/ml (2 x 10(-10) M; n = 5 experiments). This inhibitory effect was evident 48 h after the initiation of treatment and could be reversed 1 day after the cessation of TGF-beta exposure of cells. TGF-beta also reduced forskolin and (Bu)2cAMP-induced T production (greater than 85% decrease), indicating that TGF-beta can inhibit steroidogenesis distal to the formation of cAMP. The conversion of exogenously added androgen precursors (progesterone (P) and 17 alpha-hydroxyprogesterone) to T by hCG-stimulated cells was suppressed by the addition of TGF-beta. In contrast, endogenous P accumulation did not change in cultures treated with TGF-beta. Because TGF-beta-like activity has been found in the testis, the observed inhibitory effect of TGF-beta suggests a potential intratesticular regulatory role of this growth factor.
View details for Web of Science ID A1988Q278000004
View details for PubMedID 2846977
Follicle-stimulating hormone (FSH) is a glycoprotein essential for gonadal development and steroidogenesis. Recent studies suggest that deglycosylation of FSH results in the formation of antagonistic proteins that are capable of binding to gonadal receptors but that are devoid of bioactivity. Treatment of hypogonadal women with an antagonist of gonadotropin-releasing hormone substantially decreased serum FSH bioactivity with minimal changes in immunoreactivity. Chromatofocusing and size fractionation of the serum samples indicated the secretion of immunoreactive FSH isoforms that are devoid of bioactivity but that are capable of blocking FSH action in ovarian granulosa cells. These findings provide the first demonstration of naturally occurring circulating antihormones. These FSH antagonists may play an important role in the physiology and pathophysiology of the gonads.
View details for Web of Science ID A1988L435100030
View details for PubMedID 3122320
A sensitive and specific in vitro granulosa cell aromatase bioassay was adapted to measure bioactive FSH (bio-FSH) levels in urine samples. Urinary levels of bio-FSH, immunoreactive LH, estrone conjugates, and pregnanediol-3-glucuronide (PdG) were measured in first morning urine samples during the menstrual cycle in six cycling women and four lowland gorillas. The cycle length of women was relatively constant [28 +/- 1 (+/- SD) days], but varied from 28-38 days for lowland gorillas; the length of the luteal phases was relatively constant for both. All subjects had a midcycle LH peak and a luteal phase elevation in PdG. In addition, urinary estrogen excretion displayed a midcycle elevation that preceded the LH peak and a luteal phase increase similar to that of PdG. The bio-FSH levels in urine of cycling women, although at almost 100-fold higher concentrations, exhibited a pattern that closely resembled that of serum bio-FSH levels reported earlier, with an early follicular phase rise and a midcycle peak. Statistical analysis indicated a highly significant correlation (r = 0.90) between serum and urinary bio-FSH levels during the human menstrual cycle and in women in several hypo- and hypergonadotropic states, including oral contraceptive pill users, hypothalamic amenorrhea, premature ovarian failure, and postmenopause. Although a midcycle bio-FSH surge was also detected in lowland gorillas, two peaks of bio-FSH levels were consistently found during the follicular phase. The late follicular phase increase in bio-FSH levels was presumably involved in follicle selection and preceded the midcycle FSH peak by about 6 days, whereas the timing of the early follicular phase peak was variable, suggesting the involvement of complex regulatory mechanisms. These findings suggest that measurement of urinary bio-FSH levels in humans reflects serum bio-FSH in subjects in several physiological and pathological states. Studies of urinary bio-FSH levels in humans and nonhuman primates are useful in monitoring menstrual cycles, and the gorillas may be a model for understanding human reproductive cycles. The urinary granulosa cell aromatase bioassay should be useful for future assessment of bio-FSH levels in situations where serum measurements are impractical or in animal species for which specific FSH RIAs are not available.
View details for Web of Science ID A1987G176400013
View details for PubMedID 3102537
The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.
View details for Web of Science ID A1983RH09400014
View details for PubMedID 6414376