Transient, Inducible, Placenta-Specific Gene Expression in Mice
2012; 153 (11): 5637-5644
Global alteration in gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss
MOLECULAR HUMAN REPRODUCTION
2012; 18 (9): 442-450
Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues.
View details for DOI 10.1210/en.2012-1556
View details for Web of Science ID 000310359300049
View details for PubMedID 23011919
Dynamic Regulation of Wnt7a Expression in the Primate Endometrium: Implications for Postmenstrual Regeneration and Secretory Transformation
2012; 153 (3): 1063-1069
Recurrent pregnancy loss (RPL) occurs in ∼5% of women. However, the etiology is still poorly understood. Defects in decidualization of the endometrium during early pregnancy contribute to several pregnancy complications, such as pre-eclampsia and intrauterine growth restriction (IUGR), and are believed to be important in the pathogenesis of idiopathic RPL. We performed microarray analysis to identify gene expression alterations in the deciduas of idiopathic RPL patients. Control patients had one antecedent term delivery, but were undergoing dilation and curettage for current aneuploid miscarriage. Gene expression differences were evaluated using both pathway and gene ontology (GO) analysis. Selected genes were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 155 genes were found to be significantly dysregulated in the deciduas of RPL patients (>2-fold change, P < 0.05), with 22 genes up-regulated and 133 genes down-regulated. GO analysis linked a large percentage of genes to discrete biological functions, including immune response (23%), cell signaling (18%) and cell invasion (17.1%), and pathway analysis revealed consistent changes in both the interleukin 1 (IL-1) and IL-8 pathways. All genes in the IL-8 pathway were up-regulated while genes in the IL-1 pathway were down-regulated. Although both pathways can promote inflammation, IL-1 pathway activity is important for normal implantation. Additionally, genes known to be critical for degradation of the extracellular matrix, including matrix metalloproteinase 26 and serine peptidase inhibitor Kazal-type 1, were also highly up-regulated. In this first microarray approach to decidual gene expression in RPL patients, our data suggest that dysregulation of genes associated with cell invasion and immunity may contribute significantly to idiopathic recurrent miscarriage.
View details for DOI 10.1093/molehr/gas017
View details for Web of Science ID 000308243000003
View details for PubMedID 22505054
Decreased Circulating Soluble Tie2 Levels in Preeclampsia May Result from Inhibition of Vascular Endothelial Growth Factor (VEGF) Signaling
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
2011; 96 (7): E1148-E1152
Despite the vital physiological role of endometrial regeneration during the menstrual cycle and the various pathological implications of abnormal growth of endometrial epithelial cells, the local factors and regulatory mechanisms involved in endometrial regeneration and growth have not been well characterized. Here, we examine the pattern, hormone dependence, and potential functions of Wnt7a (wingless-type MMTV integration site family member 7a), which is known to play a critical role in the formation of the mouse endometrial epithelium during embryonic development, in both human and artificially cycling rhesus macaque endometrium, and using a potent Wnt-antagonist in a mouse model of endometrial regeneration. Wnt7a transcript levels were examined using quantitative real-time PCR and in situ hybridization, and immunohistochemistry was performed to detect Ki-67 and 3,5-bromodeoxyuridine. Stringent, fully conditional Wnt inhibition was achieved by adenoviral expression of Dickkopf-1 during artificial endometrial regeneration in mice. In macaques, Wnt7a expression was confined to the newly formed luminal epithelium (LE) and upper glands during the postmenstrual repair phase. The signal increased in the LE during the proliferative phase but decreased in the upper glands and was undetectable in the glands by the late proliferative phase. Interestingly, Wnt7a was completely suppressed in the LE and remained undetectable in other cell types after 7 d of progesterone treatment. The pattern of Wnt7a expression in the human endometrium was similar to that in macaques. Blockade of Wnt signaling during endometrial regeneration in mice resulted in a dramatic delay in reepithelialization and degeneration of glands and LE. These results strongly suggest, for the first time, a role for Wnt7a in postmenstrual regeneration and proliferation of endometrial glands and LE in primates, and its dramatic suppression by progesterone is likely essential for secretory transformation of the epithelium.
View details for DOI 10.1210/en.2011-1826
View details for Web of Science ID 000300645600013
View details for PubMedID 22294752
Noninvasive Monitoring of Placenta-Specific Transgene Expression by Bioluminescence Imaging
2011; 6 (1)
Recent studies have found dysregulation in circulating levels of a number of angiogenic factors and their soluble receptors in preeclampsia. In this study, we examined the mechanism of production of soluble Tie2 (sTie2) and its potential connection to the failure of vascular remodeling in preeclamptic pregnancies.Serum samples were collected prospectively from 41 pregnant subjects at five different time points throughout pregnancy. Five of these subjects developed preeclampsia. For a second study, serum and placental samples were collected at delivery from preeclamptic and gestational age-matched controls. We examined serum sTie2 levels, and angiopoietin 1, angiopoietin 2, and Tie2 mRNA expression and localization in placental samples from the central basal plate area. We also examined the effects of vascular endothelial growth factor (VEGF) and a matrix metalloproteinase (MMP) inhibitor on proteolytic shedding of Tie2 in uterine microvascular endothelial cells.Serum sTie2 levels were significantly lower in preeclamptic subjects starting at 24-28 wk of gestation and continued to be lower through the time of delivery. In culture experiments, VEGF treatment significantly increased sTie2 levels in conditioned media, whereas the MMP inhibitor completely blocked this increase, suggesting that VEGF-induced Tie2 release is MMP dependent.Our data suggest, for the first time, an interaction between VEGF and Tie2 in uterine endothelial cells and a potential mechanism for the decrease in circulating sTie2 levels in preeclampsia, likely through inhibition of VEGF signaling. Further studies on VEGF-Tie2 interactions during pregnancy should provide new insights into the mechanisms underlying the failure of vascular remodeling in preeclampsia and other pregnancy complications.
View details for DOI 10.1210/jc.2011-0063
View details for Web of Science ID 000292454500015
View details for PubMedID 21525162
Adam12 plays a role during uterine decidualization in mice
CELL AND TISSUE RESEARCH
2009; 338 (3): 413-421
Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts.Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2)/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages.These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero.
View details for DOI 10.1371/journal.pone.0016348
View details for Web of Science ID 000286522300037
View details for PubMedID 21283713
View details for PubMedCentralID PMC3025029
CXCL14 Inhibits Trophoblast Outgrowth Via a Paracrine/Autocrine Manner During Early Pregnancy in Mice
JOURNAL OF CELLULAR PHYSIOLOGY
2009; 221 (2): 448-457
In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.
View details for DOI 10.1007/s00441-009-0884-9
View details for Web of Science ID 000271978700008
View details for PubMedID 19841944
VEGF blockade inhibits angiogenesis and reepithelialization of endometrium
2008; 22 (10): 3571-3580
CXCL14, a member of chemokine family, was previously known to participate in many pathophysiological events, such as leukocytes recruitment and tumor suppression. However, it remained largely unknown whether CXCL14 is a physiological player during early pregnancy. In this regard, our recent global gene microarray analysis has observed an implantation-specific expression profile of CXCL14 mRNA during early pregnancy in mice, showing its higher levels at implantation sites compared to inter-implantation sites, implicating a potential role of CXCL14 in the periimplantation events. In the present investigation, using Northern blot, in situ hybridization and immunostaining, we further demonstrated that uterine CXCL14 expression was specifically induced at embryo implantation site and expanded with subsequent decidualization process in a spatiotemporal manner. The implanting embryo also showed a highlighted expression of CXCL14 in the blastocyst trophectoderm and its derived ectoplacental cones (EPCs) during postimplantation development. In vitro functional study revealed that CXCL14 could significantly inhibit both primary and secondary trophoblast attachment and outgrowth, correlated with a stage-dependant downregulation of MMP-2 and/or MMP-9 activity. Moreover, it was found that biotinylated CXCL14 could specifically bind to trophoblast cells in vitro and in vivo, suggesting trophoblast cell, perhaps expressing the unidentified CXCL14 receptor, is a bioactive target of CXCL14. Collectively, our findings provide evidences supporting the contention that CXCL14 is an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal-fetal interface during the process of pregnancy establishment. This study is clinically related since CXCL14 is also highly expressed in human receptive endometrium and trophoblasts.
View details for DOI 10.1002/jcp.21877
View details for Web of Science ID 000270438300023
View details for PubMedID 19626669
Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity
CELL BIOLOGY INTERNATIONAL
2007; 31 (1): 24-29
Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangiogenesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2-neuropilin 1 (VEGFR2-NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endometrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angiogenesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/postpartum repair.
View details for DOI 10.1096/fj.08-111401
View details for Web of Science ID 000259642600019
View details for PubMedID 18606863
Dickkopf-1 induced apoptosis in human placental choriocarcinoma is independent of canonical Wnt signaling
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2006; 350 (3): 641-647
Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P<0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P<0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.
View details for DOI 10.1016/j.cellbi.2006.09.003
View details for Web of Science ID 000244297300004
View details for PubMedID 17052925
Inhibition of the beta-catenin signaling pathway in blastocyst and uterus during the window of implantation in mice
BIOLOGY OF REPRODUCTION
2005; 72 (3): 700-706
Placental choriocarcinoma, a reproductive system carcinoma in women, has about 0.81% occurrence frequency in China, which leads to over 90% lethality due to indistinct pathogenesis and the absence of efficient therapeutic treatment. In the present study, using immunostaining and reverse transcription PCR, we reported that Dickkopf-1 (Dkk-1) is prominently expressed in human cytotrophoblast (CTB) cell, but absent in the human placental choriocarcinoma cell line JAR and JEG3, implicating an unknown correlation between Dkk-1 and carcinogenesis of placental choriocarcinoma. Further, through exogenous introduction of Dkk-1, we found repressed proliferation in JAR and JEG3, induced apoptosis in JAR, and discovered significant tumor suppression effects of Dkk-1 in placental choriocarcinoma. Moreover we found that this function of Dkk-1 is achieved through c-Jun N-terminal kinase (JNK), whereas the canonical Wnt pathway may not have a great role. This discovery is not symphonic to previous functional understanding of Dkk-1, a canonical Wnt signaling antagonist. Together, our data indicate the possible correlation between Dkk-1 and human placental choriocarcinoma and suggest potential applications of Dkk-1 in treatment of human placental choriocarcinomas.
View details for DOI 10.1016/j.bbrc.2006.09.087
View details for Web of Science ID 000241584300023
View details for PubMedID 17026960
Beta-catenin, the mammalian homolog of Drosophila armadillo protein, was first identified as a cadherin-associated protein at cell-cell junctions. Another function of beta-catenin is the transduction of cytosolic signals to the nucleus in a variety of cellular contexts, which usually are elicited by the active form of beta-catenin. The aim of the present study was to examine the potential role of active beta-catenin in the mouse embryo and uterus during embryo implantation. Active beta-catenin was detected differentially in mouse embryos and uteri during the peri-implantation period. Aberrant activation of beta-catenin by LiCl, a well-known glycogen synthase kinase-3 inhibitor, significantly inhibited blastocyst hatching and subsequent adhesion and outgrowth on fibronectin. Results obtained from pseudopregnant and implantation-delayed mice imply an important role for implanting blastocysts in the temporal and spatial changes of active beta-catenin in the uterus during the window of implantation. Collectively, these results suggest that the beta-catenin signaling pathway is inhibited in both blastocyst and uterus during the window of implantation, which may represent a new mechanism to synchronize the development of preimplantation embryos and differentiation of the uterus during this process.
View details for DOI 10.1095/biolreprod.104.033837
View details for Web of Science ID 000227296400024
View details for PubMedID 15496516