Independent Studies (3)
- Advanced Research Laboratory in Experimental Biology
BIO 199 (Sum)
- Directed Reading in Biology
BIO 198 (Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Sum)
- Advanced Research Laboratory in Experimental Biology
- Efflux transporters: Newly appreciated roles in protection against pollutants ENVIRONMENTAL SCIENCE & TECHNOLOGY 2008; 42 (11): 3914-3920
Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus
2007; 303 (1): 336-346
Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.
View details for DOI 10.1016/j.ydbio.2006.11.018
View details for Web of Science ID 000244542800028
View details for PubMedID 17174294
Embryo stability and vulnerability in an always changing world
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (6): 1745-1750
Contrary to the view that embryos and larvae are the most fragile stages of life, development is stable under real-world conditions. Early cleavage embryos are prepared for environmental vagaries by having high levels of cellular defenses already present in the egg before fertilization. Later in development, adaptive responses to the environment either buffer stress or produce alternative developmental phenotypes. These buffers, defenses, and alternative pathways set physiological limits for development under expected conditions; teratology occurs when embryos encounter unexpected environmental changes and when stress exceeds these limits. Of concern is that rapid anthropogenic changes to the environment are beyond the range of these protective mechanisms.
View details for DOI 10.1073/pnas.0610108104
View details for Web of Science ID 000244127900005
View details for PubMedID 17264211
New perspectives on perfluorochemical ecotoxicology: inhibition and induction of an efflux transporter in the marine mussel, Mytilus californianus
ENVIRONMENTAL SCIENCE & TECHNOLOGY
2006; 40 (17): 5580-5585
The toxicological effects of perfluoroalkyl acids on the p-glycoprotein (p-gp) cellular efflux transporter were investigated using the marine mussel Mytilus californianus as a model system. Four of the perfluoroalkyl acids studied exhibit chemosensitizing behavior, significantly inhibiting p-gp transporter activity. The inhibitory potency is maximal for the longer chain acids perfluorononanoate (PFNA) and perfluorodecanoate (PFDA), with average IC50 values of 4.8 and 7.1 microM, respectively. Results indicate that PFNA inhibits p-gp by an indirect mechanism, and this inhibition is reversible and accompanied by a rapid loss of PFNA from the tissue. In addition, PFNA induces expression of the p-gp transporter after a 2-h exposure, a stress response that may result in a metabolic cost to the organism. Given that most organisms, including humans, share efflux transporters as a first line of defense against toxicants, the results of this study may have broader implications for the ecotoxicology of perfluoroalkyl acids.
View details for DOI 10.1021/es0602593
View details for Web of Science ID 000240130200067
View details for PubMedID 16999143
Nitromusk and polycyclic musk compounds as long-term inhibitors of cellular xenobiotic defense systems mediated by multidrug transporters
ENVIRONMENTAL HEALTH PERSPECTIVES
2005; 113 (1): 17-24
Synthetic musk compounds, widely used as fragrances in consumer products, have been detected in human tissue and, surprisingly, in aquatic organisms such as fish and mollusks. Although their persistence and potential to bioaccumulate are of concern, the toxicity and environmental risks of these chemicals are generally regarded as low. Here, however, we show that nitromusks and polycyclic musks inhibit the activity of multidrug efflux transporters responsible for multixenobiotic resistance (MXR) in gills of the marine mussel Mytilus californianus. The IC(subscript)10(/subscript) (concentration that inhibits 10%) values for the different classes of musks were in the range of 0.09-0.39 microM, and IC(subscript)50(/subscript) values were 0.74-2.56 microM. The immediate consequence of inhibition of efflux transporters is that normally excluded xenobiotics will now be able to enter the cell. Remarkably, the inhibitory effects of a brief 2-hr exposure to musks were only partially reversed after a 24- to 48-hr recovery period in clean seawater. This unexpected consequence of synthetic musks--a long-term loss of efflux transport activity--will result in continued accumulation of normally excluded toxicants even after direct exposure to the musk has ended. These findings also point to the need to determine whether other environmental chemicals have similar long-term effects on these transporters. The results are relevant to human health because they raise the possibility that exposure to common xenobiotics and pharmaceuticals could cause similar long-term inhibition of these transporters and lead to increased exposure to normally excluded toxicants.
View details for Web of Science ID 000226196300024
View details for PubMedID 15626642
Activation of multidrug efflux transporter activity at fertilization in sea urchin embryos (Strongylocentrotus purpuratus)
2004; 276 (2): 452-462
This study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine, confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions.
View details for DOI 10.1016/j.ydbio.2004.09.013
View details for Web of Science ID 000225777500017
View details for PubMedID 15581878
Phosphoinositide metabolism at fertilization of sea urchin eggs measured with a GFP-probe
DEVELOPMENT GROWTH & DIFFERENTIATION
2004; 46 (5): 413-423
Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.
View details for Web of Science ID 000225322600003
View details for PubMedID 15606487
Some precautions in using chelators to buffer metals in biological solutions
2004; 35 (5): 427-431
Chelators and associated computer programs are commonly used to buffer metal ions in biological experiments. This communication discusses common misunderstandings and pitfalls in use of these buffers and provides information on choosing the best metal buffer for different experimental situations.
View details for DOI 10.1016/j.ceca.2003.10.006
View details for Web of Science ID 000220483300004
View details for PubMedID 15003852
- Sea urchin gametes in the teaching laboratory: Good experiments and good experiences DEVELOPMENT OF SEA URCHINS, ASCIDIANS, AND OTHER INVERTEBRATE DEUTEROSTOMES: EXPERIMENTAL APPROACHES 2004; 74: 797-823
Nitric oxide in oocyte maturation, ovulation, fertilization, cleavage and implantation: A little dab'll do ya
CURRENT PHARMACEUTICAL DESIGN
2003; 9 (5): 399-409
Nitric oxide synthases, the enzymes that generate NO gas, may be involved in reproduction and development of multicellular organisms at many levels and thus provide important targets for design of drugs to intervene in reproductive processes. This review focuses on the role of nitric oxide in key events of reproduction including gamete activation, fertilization, early cell divisions and implantation. A general trend highlighted by the studies reviewed is that NO plays a biphasic role in reproduction. That is, a narrow range of NO concentrations, usually low, will stimulate or enhance these early events in reproduction, but either a lack of NO or too much NO has negative consequences. One of the shortcomings of the field currently is the lack of molecular detail concerning the mechanism of NO action. This has been due in part to lack of technology for effective detection of NO and its molecular targets. A few targets of NO have been indirectly implicated and advances in this area of research will provide substrates for development of drugs to control reproductive function. Work from both invertebrate and vertebrate model systems is presented and implications for control of reproductive physiology discussed. Ubiquity of NO signaling in animals may mean that effective control of reproduction must target mediators of NO action and not NOS enzymes themselves.
View details for Web of Science ID 000180329900005
View details for PubMedID 12570817
Protection of DNA during early development: adaptations and evolutionary consequences
EVOLUTION & DEVELOPMENT
2003; 5 (1): 83-88
The rapidly dividing cleavage stages of embryos do not have the typical responses to cell damage, such as induction of the heat shock response, use of mitotic checkpoints, or use of apoptosis to eliminate severely damaged cells. This could create problems with integrity of DNA, but the solution in these embryos appears to be a "be prepared" approach, in which specific adaptations are used to minimize DNA damage during cleavage and the use of apoptosis at the mid-blastula transition to remove any cells that were nevertheless damaged. It has been assumed that this approach has evolved because of the advantage of rapid production of a motile larvae. Alternatively, this particular approach may have the selective advantage of increasing mutation rate when there are greater environmental stresses. This could provide more variants on which selective pressures could act and thus accelerate evolution during environmentally stressful periods.
View details for Web of Science ID 000179925400013
View details for PubMedID 12492414
NO is necessary and sufficient for egg activation at fertilization
2000; 406 (6796): 633-636
The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.
View details for Web of Science ID 000088653800049
View details for PubMedID 10949304
Induction of the multixenobiotic defense mechanism (MXR), P-glycoprotein, in the mussel Mytilus californianus as a general cellular response to environmental stresses.
Aquatic toxicology (Amsterdam, Netherlands)
2000; 49 (1-2): 89-100
A multixenobiotic resistance mechanism (MXR) related to the P-glycoprotein multidrug transporter protein (p-gp) has been identified and characterized in several marine invertebrates. p-gp activity and protein titer is induced by exposure to toxins, supporting the suggestion that the role for this transporter is protection from xenobiotics by reducing accumulation of toxins in cells. In this study, we report on the specificity of the induction of the transporter by various chemical and physical stressors. p-gp substrates (including the pesticides pentachlorophenol and chlorthal) as well as non-substrates (including DDE and sodium arsenite) induced p-gp activity and protein titer in the gill tissues of the mussel Mytilus californianus. Similarly, mussels exposed to heat shock of 20 degrees C or 25 degrees C exhibited increased p-gp titer and activity compared to mussels held at ambient (12 degrees C) temperature seawater. Some of the same treatments that induced an increase in p-gp caused a concomitant increase in hsp70, but hsp induction was not always associated with induction of the p-gp protein. These findings suggest that p-gp induction in mussels may be part of a general cellular stress response. This response, however, does not appear to be always coupled with the hsp70 response in mussels.
View details for PubMedID 10814809
The roles of changes in NADPH and pH during fertilization and artificial activation of the sea urchin egg
1999; 216 (1): 394-405
Incubating unfertilized sea urchin eggs in weak bases activates nuclear centering, DNA synthesis, and chromosome cycles. These effects were initially attributed to raising the intracellular pH (pH(i)), but later experiments indicated that these weak bases also lead to increases in reduced pyridine nucleotides. These findings raised the question whether the activation of the nucleus was due to increased pH(i) or to increased NAD(P)H or possibly other effects. This report attempts to clarify how ammonia activates eggs by independently altering NADPH and pH(i). To increase the pH(i), unfertilized eggs were injected with zwitterionic buffers. This stimulated pronuclear centering, DNA synthesis, and nuclear envelope breakdown; there appeared to be a threshold corresponding to the fertilized pH(i). However, like incubation in ammonia, injection of base also increased NAD(P)H. The NAD(P)H rise caused by directly raising the pH(i) occurred in the presence of intracellular calcium chelators, indicating that calcium is not required. Increasing NAD(P)H alone did not activate nuclear centering, DNA synthesis, or nuclear envelope breakdown. Although these experiments cannot eliminate a role for the NADPH increase in initiating events leading to nuclear centering and entry into mitosis, they provide additional and strong evidence that increasing the pH(i) may be a primary signal.
View details for Web of Science ID 000084171500030
View details for PubMedID 10588888
Redox changes during fertilization and maturation of marine invertebrate eggs
1998; 203 (1): 1-11
Previous studies on sea urchin eggs indicate that activation of NAD kinase is one of the earliest Ca2+-mediated events of fertilization. The subsequently produced NADP is converted to NADPH by the pentose shunt pathway, and some of this NADPH is used by an NADPH oxidase for generation of H2O2. To examine whether these changes apply generally, we have analyzed changes in pyridine nucleotide content during meiotic maturation and fertilization in eggs from four phyla. Surprisingly, fertilization-associated increases in NAD kinase were found only in echinoid eggs. The ratio of NADPH/NADP (redox ratio) increased from 1-1.6 to 2.5-6 following fertilization of echinoid and also clam eggs. However, the ratio is already >2 for unfertilized asteroid, tunicate and echiuroid eggs, and this ratio is unaffected by fertilization. We conclude that activation of NAD kinase and shifts in pyridine nucleotide metabolism and thereby cellular redox status may have roles that vary between species. In echinoids, a major role is in providing NADPH for H2O2 production, but there may be other yet unappreciated signaling functions for this change.
View details for Web of Science ID 000076869400001
View details for PubMedID 9806768
- Daniel Mazia: a passion for understanding how cells reproduce TRENDS IN CELL BIOLOGY 1998; 8 (10): 416-418
Effects of wheat germ agglutinin on tunicate egg activation and fertilization: Is there a plasma membrane sperm receptor system on Ascidia ceratodes eggs?
DEVELOPMENT GROWTH & DIFFERENTIATION
1998; 40 (3): 297-306
Little work has been carried out on the sperm recognition systems present on the egg plasma membrane. Here it is shown that wheat germ agglutinin (WGA) interferes with the sperm-interacting system on the plasma membrane of eggs of the ascidian, Ascidia ceratodes. The WGA activates the dechorionated egg, indicating that a plasma membrane sugar residue can be directly tied to egg activation. Low concentrations of this lectin do not activate the eggs, but reduce fertilizability. This observation suggests that the WGA binding site might be part of a sperm reception-activation complex in the plasma membrane. While WGA also affects sperm binding to the chorion, the mechanisms of sperm interaction at the plasma membrane and chorion show different sensitivities to lectins, sugars and enzymes.
View details for Web of Science ID 000074009800005
View details for PubMedID 9639357
THE IN-VIVO RATE OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE ACTIVITY IN SEA-URCHIN EGGS DETERMINED WITH A PHOTOLABILE CAGED SUBSTRATE
1995; 169 (2): 733-744
Some of the earliest metabolic changes after fertilization of sea urchin eggs center around the activity of the pentose phosphate shunt. We here report on the in vivo activity of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of this shunt, as assayed with a photolabile (caged) analog of the substrate, glucose-6-phosphate (G6P). Caged G6P was synthesized from radiolabeled (5-3H or 1-14C) glucose and loaded into unfertilized sea urchin eggs by transient electroporation. Irradiation of these eggs (either before or after fertilization) photolyses the caged G6P, thereby pulsing the cell with 3H- and 14C-labeled G6P. The fluxes of G6P into glycolysis and the pentose shunt are calculated from the rates of oxidation of labeled G6P to 3H2O and 14CO2; since the turnover of the 6-phosphogluconate pool by 6-phosphogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and Epel, D. (1992) Exp. Cell Res. 201:366-372), the rate of 14CO2 production by the pentose shunt is equal to the flux of G6P through G6PDH. The data indicate that G6PDH activity is very low in unfertilized eggs, increases 184- to 427-fold by 2 min after fertilization, and then decreases to a value that is 74 to 209 times the unfertilized level (maximally 0.005 x 10(-8) units per egg in unfertilized eggs, 2.14 x 10(-8) units per egg by 2 min after fertilization, and 1.05 x 10(-8) units per egg by 20 min after fertilization). In spite of this substantial activation, the enzyme activity is considerably repressed; compared with activity in broken cell extracts, G6PDH at these developmental times operates in vivo at 0-0.003%, 0.52-1.21%, and 0.21-0.59%, respectively, of its potential activity. These results are discussed in terms of various hypotheses regarding the modulation of G6PDH activity by fertilization. These activity measurements relate well to other indices of in vivo activity. The major use of the NADPH shortly after fertilization is to produce H2O2, which is used as a substrate for fertilization membrane hardening; our data indicate that the NADPH that is produced by the pentose shunt activity is 30-70% of that required for this postfertilization generation of H2O2.
View details for Web of Science ID A1995RD48300030
View details for PubMedID 7781912
PROTEIN-SYNTHESIS INCREASES AFTER FERTILIZATION OF SEA-URCHIN EGGS IN THE ABSENCE OF AN INCREASE IN INTRACELLULAR PH
1995; 169 (2): 683-698
We have reevaluated the presumed requirement for an elevated intracellular pH (pHi) in the acceleration of protein synthesis which follows fertilization of eggs of the sea urchin Lytechinus pictus. Zygotes were transferred to sea water at a low pH (6.8) containing a permeant weak acid at times ranging from 5 min to as early as 30 sec postinsemination, to reverse or prevent the rise in pHi that normally ensues upon fertilization. Using the fluorescent pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we show that transfer of zygotes at 1 min or earlier after fertilization essentially blocks the rise in pHi. Under these conditions, both the incorporation of radiolabeled leucine into protein and the assembly of ribosomes into polysomes increase substantially (> 50% of control values). We also assessed leucine incorporation during incubation of eggs and zygotes in sodium-free sea water or sea water containing amiloride, two additional treatments that block the pHi rise. In the presence of amiloride, leucine incorporation increased upon fertilization, whereas little or no increase was observed in sodium-free sea water. We provide evidence that the low rates of leucine incorporation in sodium-free sea water result from the tendency for this experimental condition to lower the pHi to values significantly lower than the pHi in unfertilized eggs. These findings call into doubt the belief that the pHi rise at fertilization is a necessary prerequisite for the acceleration of bulk protein synthesis. These observations support the view that pHi is only one of several signals involved in the turn on of protein synthesis at the time of fertilization of sea urchin eggs.
View details for Web of Science ID A1995RD48300026
View details for PubMedID 7781908
BEAKERS VERSUS BREAKERS - HOW FERTILIZATION IN THE LABORATORY DIFFERS FROM FERTILIZATION IN NATURE
1995; 3 (2): 95-99
The fertilisation of free-spawning invertebrates, mainly sea urchins, has been studied extensively during the last hundred years. However, results obtained from in vitro experiments do not always reflect what happens in the real world. Organisms in their natural habitats have a complex set of challenges, cues and behaviours to contend with during fertilisation and early development, factors that are normally not considered in the laboratory setting. This review examines recent work on fertilisation ecology and discusses the relevance of these results to the findings gleaned from laboratory research. Emphasis is placed on stresses associated with fertilisation in situ, and how responses to environmental stresses (such as from turbulence, oxidative stress, ultraviolet radiation and pathogens) might affect the fertilisation process.
View details for Web of Science ID A1995RB56400001
View details for PubMedID 7582921
THE USE OF CAGED SUBSTRATES TO ASSESS THE ACTIVITY OF 6-PHOSPHOGLUCONATE DEHYDROGENASE IN LIVING SEA-URCHIN EGGS
EXPERIMENTAL CELL RESEARCH
1992; 201 (2): 366-372
As part of our inquiries into the regulation of the hexose monophosphate shunt in the early development of sea urchin eggs and embryos, we have developed a novel method to assess the in vivo activity of the enzyme 6-phosphogluconate dehydrogenase (6PGDH) before and after fertilization. Our measurements show that the intracellular level of 6-phosphogluconate (6PG) in eggs decreases 60% after fertilization, which is consistent with the increase in the activity of 6PGDH previously reported using irreversibly permeabilized cell assays (Swezey and Epel, Proc. Natl. Acad. Sci USA 85, 812-816, 1988). The in vivo turnover of the 6PG pool was assessed using a new radioisotopic technique. 1-14C-labeled 6PG was chemically modified such that it was not metabolized by cellular 6PGDH and could be rapidly converted back to 6PG by photolysis. This "caged" 6PG was introduced into unfertilized sea urchin eggs using a transient permeabilization procedure, and then the oxidation of [1-14C]6PG in vivo upon irradiation was followed. Oxidation of 6PG was complete within 7-11 s of irradiation, indicating an extremely rapid turnover of this pool in sea urchin eggs. Based on the 6PG pool sizes and the kinetic properties of 6PGDH, determined here, along with the activity levels seen in permeabilized cells, the half-time for the label in the 6PG pool in sea urchin eggs is calculated to be 26 s. This is inconsistent with the in vivo turnover rates seen in these studies, indicating that the permeabilized cell assays overestimate the degree of inhibition of 6PGDH before fertilization. These results suggest that caution should be exercised in extrapolating data obtained from permeabilized cells to the situation in vivo.
View details for Web of Science ID A1992JH39000015
View details for PubMedID 1639134
INVIVO PROTEIN-PHOSPHORYLATION AND LABELING OF ATP IN SEA-URCHIN EGGS LOADED WITH PO4-P-32 VIA ELECTROPORATION
1991; 148 (1): 156-164
Protein phosphorylation was examined in sea urchin eggs in which the ATP was labeled with 32P over a brief period of time using reversible electrical poration to gain access to the cytoplasm. Unfertilized eggs from two species, Lytechinus pictus and Strongylocentrotus purpuratus, were electrically permeabilized and incubated in the presence of [32P]H3PO4, under conditions allowing label uptake. After a 5-min loading period the eggs were resealed and the fate of the label was monitored. The label had equilibrated with the cellular ATP pool within the 13-min period required for loading and resealing the eggs. Furthermore, this equilibrium was maintained for at least 2 hr beyond the loading period in either unfertilized or fertilized eggs (i.e., the specific activity of ATP was the same for fertilized and unfertilized eggs). We also examined the position of the label within the ATP and found that 40-45% of the label isolated with the ATP was in the gamma phosphate of ATP and hence was immediately available for protein phosphorylation. The label was maintained in this position in the ATP for at least 2 hr following the loading period and was not affected by fertilization (determined for L. pictus only). The phosphoprotein banding pattern was determined by gel electrophoresis and autoradiography at various time points following the loading period. There was a continuous increase of label incorporated into protein over time; however, the banding pattern did not change. This pattern was not affected by fertilization. Furthermore, inhibition of protein synthesis (with emetine) had no effect on this phosphoprotein banding pattern. Although the loading period was brief there was sufficient incorporation of label into protein during this time to obscure potential regulatory phosphorylation events.
View details for Web of Science ID A1991GP35800015
View details for PubMedID 1936555
THE INITIATION OF DEVELOPMENT AT FERTILIZATION
CELL DIFFERENTIATION AND DEVELOPMENT
1990; 29 (1): 1-12
As seen, important advances have now been made in understanding the beginning of development at fertilization. Free calcium and pHi level changes result from a sperm-mediated breakdown of PPI with production of IP3. The resultant calcium increase, either alone or in concert with diacylglycerol, activates the Na(+)-H+ exchange and a consequent cytosolic pHi level increase. The calcium increase is responsible for the NADP change (via NAD kinase) and possibly the change in G6PD. These two changes could be involved solely in producing NADPH for fertilization membrane hardening or these changes could also have some role in the later initiation of DNA synthesis. The finding that other enzymes assayed in permeabilized cells also evince large changes in activity suggests that a global change may be occurring with important portents for cell activity. The role of calcium in furthering subsequent synthetic events, however, is unclear since no calcium target has yet been described that is necessary for the subsequent specific synthesis of proteins, as cyclins, or for the initiation of DNA synthesis. The pHi level increase, in concert with increased calcium, might be sufficient to start off protein synthesis and subsequent cyclin accumulation. However, the pHi level increase, independently of protein synthesis, can initiate new DNA synthesis. These independent events converge in the putative activation of MPF by cyclin, which then starts off the first mitotic cycle. Other independent events are associated with the sperm entry, cortical modifications, fertilization membrane elevation and the numerous changes leading to the fusion of the sperm and egg nucleus in the egg center. Fertilization represents one of the best studied examples of how a covert developmental program is made overt by an external messenger. The challenges for the near future are to explain how sperm-egg contact leads to PPI hydrolysis and how pHi level changes (and Cai level changes?) lead to the initiation of the cell cycle. The challenge for the distant future is describing how this program is set up during oogenesis.
View details for Web of Science ID A1990CN64900001
View details for PubMedID 2154300
STABLE, RESEALABLE PORES FORMED IN SEA-URCHIN EGGS BY ELECTRIC-DISCHARGE (ELECTROPORATION) PERMIT SUBSTRATE LOADING FOR ASSAY OF ENZYMES INVIVO
1989; 1 (1): 65-74
We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.
View details for Web of Science ID A1989DM09500007
View details for PubMedID 2519619
THE LOCALIZATION OF PI AND PIP KINASE-ACTIVITIES IN THE SEA-URCHIN EGG AND THEIR MODULATION FOLLOWING FERTILIZATION
1989; 131 (1): 236-242
Phosphatidylinositol phosphate (PIP) kinase activity is localized to the cortical region of unfertilized sea urchin eggs, while phosphatidylinositol (PI) kinase activity is found in both cortical and noncortical membranes. Following fertilization PIP kinase activity decreases, while PI kinase activity remains unchanged. The selective loss of PIP kinase activity is related to cortical granule exocytosis since the drop in activity does not occur if exocytosis is prevented by high hydrostatic pressure. When isolated cortices are exposed to elevated concentrations of calcium, both the PI and PIP kinase activities increase, suggesting that activation of these enzymes might occur when calcium levels increase within the fertilized egg prior to cortical granule exocytosis. The polyamine spermine also stimulates the formation of phosphatidylinositol bisphosphate at physiological concentrations.
View details for Web of Science ID A1989R528900023
View details for PubMedID 2535822
ENZYME STIMULATION UPON FERTILIZATION IS REVEALED IN ELECTRICALLY PERMEABILIZED SEA-URCHIN EGGS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (3): 812-816
Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity. At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells. Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions. This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells. The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 220.127.116.11) in permeabilized cells was studied in greater detail and has the following characteristics. (i) Regulation of activity persists during early development. (ii) This regulation is not mediated by diffusible allosteric agents. (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization. (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs. activity profiles. These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.
View details for Web of Science ID A1988L992200036
View details for PubMedID 3422463