THE JOURNAL OF EXPERIMENTAL ZOOLOGY 262~391-404(1992)

Modulation of Meiotic Arrest in Mouse Oocytes by Guanyl Nucleotides and Modifiers of G-Proteins STEPHEN M. DOWNS, ROBERTO BUCCIONE, AND JOHN J . EPPIG Biology Department, Marquette Uniuersity, Milwaukee, Wisconsin 53233 (S.M.D.) and The Jackson Laboratory, Bar Harbor, Maine 04609 (R.B., J .J.E.) ABSTRACT Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in CAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTPyS. This effect was specific for GTPyS, because GppNHp, GTP, and ATPyS were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF, 1. Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF, abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF, alone were without effect. Both cholera toxin and A1F4- augmented the stimulatory action of FSH on CAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved a t the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the cx subunit of G, and increased generation of CAMP,whereas AlF, appears to act by antagonizing a CAMP-dependentstep. o 1992 WiIey-Liss, Inc. ~

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Guanyl nucleotide-binding-proteins (G-proteins) participate in a wide variety of biological processes, particularly those that require the coupling of an external stimulus to an effector system. A family of membrane-associated, heterotrimeric G-proteins are comprised of a,p, and y subunits, in order of decreasing size. When appropriate ligands bind to specific receptors, these G-proteins bind GTP, become activated, and subsequently associate with, and thereby alter the activity of, an effector. Cyclic adenosine monophosphate (CAMP)production in several systems is catalyzed by the activity of membrane-bound adenylate cyclase upon association of the GTP-bound, active a subunit of G, (stimulatory G-protein) with the enzyme. Hydrolysis of GTP to GDP by the a subunit then terminates activation of the enzyme by favoring reassociation with the py dimer (Birnbaumer et al., '85; Gilman, '87; Neer and Clapham, '88). Since cAMP is probably a n 0 1992 WILEY-LISS.INC.

important regulator of oocyte maturation (Cho et al., '74; Wassarman et al., '76; Downs et al., '88), it is likely that G-proteins participate in the regulation of this physiological process, although this participation has not been directly demonstrated. There is considerable evidence that G-proteins play a pivotal role in the regulation of oocyte maturation in frogs and starfish. For example, microinjection of the a subunit of Gointo Xenopus oocytes promotes germinal vesicle breakdown (GVB;Kroll et al., '90). In addition, a small GTP-binding, ADPribosylation factor (ARF) prevents both insulin- and progesterone-stimulated GVB in Xenopus oocytes (Bahnson et al., '89).R a s p21 proteins, which share homology with the G-protein a subunit and also Received March 1,1991; revision accepted December 2, 1991. Roberto Buccione is now at Consorzio Mario Negri Sud, Centro di Ricerche Farmacologiche e Biomediche, 66030 S. Maria Imbaro, Chieti, Italy.

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bind GTP, stimulate GVB when microinjected into frog oocytes (Birchmeier et al., '85);moreover, antibody to this protein suppresses insulin-stimulated GVB (Deshpande and Kung, '87; Korn et al., '87) and augments the stimulatory action of progesterone (Sadler et al., '86). The cholera and pertussis bacterial toxins, which bind to and ADP-ribosylate G-protein a subunits and modify their behavior, have also been shown to influence the oocyte's maturational state. The microinjected A protomer of pertussis toxin prevents hormone-mediated GVB in Xenopus oocytes (Pellaz and Shorderet-Slatkine, '89), and pertussis toxin has a similar effect in 1-methyladeninine-treatedstarfish oocytes (Shilling and Jaffe, '87). Further, a nonhydrolyzable analog of GDP, GDP,S, interferes with the induction of meiotic maturation when microinjected into starfish oocytes (Shilling and Jaffe, '87). Some evidence has implicated guany1compounds in the control of meiotic maturation in mammalian oocytes. When various purines were tested for their ability t o suppress spontaneous oocyte maturation in vitro, guanine and guanosine proved to be the most potent (Downs et al., '85).In addition, mycophenolic acid and bredinin, inhibitors of inosine monophosphate (IMP) dehydrogenase, promoted the resumption of meiosis either when added to culture medium in which oocytes were maintained in meiotic arrest with hypoxanthine (Downs et al., '86) or when administered by intraperitoneal injection to pregnant mares' serum gonadotropin (PMSGkprimed mice (Downs and Eppig, '87). Since IMP dehydrogenase catalyzes the first reaction in a two-step pathway from IMP to GMP, the results support a role for guanyl compounds in the maintenance of meiotic arrest. Guanosine has been shown to be a potent inhibitor of cAMP phosphodiesterase in oocyte lysates and thus could prevent oocyte maturation by inhibiting catabolism of cAMP (Downs et al., '89). However, an alternative mechanism by which guanyl compounds might participate in the maintenance of meiotic arrest is through phosphorylation to GTP; this nucleotide could then interact with G-proteins to influence the release of various second messengers, perhaps CAMP,involved in maintaining the immature, GV stage (Downs, '90b). An inhibitory action of cholera toxin on the spontaneous maturation of mouse oocytes (Vivarelli et al., '83; Downs and Eppig, '85) is consistent with this idea. The interaction of guanyl nucleotides with G-proteins within the somatic follicle cells could also be important in the generation of mediators in response to preovulatory gonadotropin stimulation that provoke the resump-

tion of meiotic maturation. Furthermore, immunocytochemistry studies have demonstrated the presence of G-protein Py dimer at the plasma membrane of rat oocytes, supporting the concept that G-proteinsparticipate in oocyte development or function (Garty et al., '88). The present experiments were carried out to examine the possible involvement of G-proteins in the control of mouse oocyte maturation. We have used two approachesto study this question. The first involved microinjection of nucleotides and nucleotide analogs into denuded oocytes and assaying the oocytes for GVB after an appropriate incubation period. The second approach examined the effect of compounds known to modulate the activity of G-proteins on both spontaneous and hormone-induced oocyte maturation. The data support the idea that G-proteinsplay a critical role in both the maintenance of meiotic arrest and the hormonal stimulation of the resumption of maturation.

MATERIALS AND METHODS Oocyte isolation and culture system For all experiments, 19-21-day-old (C57BL/6J X SJL/J) F1 mice were used. Animals received 5 IU PMSG, were killed 48 h later, and the ovaries were removed and placed in medium supplemented (in most experiments) with 0.2 mM 3-isobutyl-lmethylxanthine (IBMX).This drug, a cAMP phosphodiesterase inhibitor, maintained meiotic arrest during the isolation procedure. Antral follicleswere poked with sterile 25-gauge needles and the released cumulus cell-enclosed oocytes were collected with a micropipet. For experiments involving denuded oocytes, cumulus cell-enclosed oocytes were drawn in and out of a manually operated Pasteur pipet to remove cumulus cells. For the microinjection experiments, denuded oocytes were microinjected while maintained in meiotic arrest with the isolation medium. Cumulus cell-enclosed oocytes or denuded oocytes were washed through four changes of IBMX-free medium and transferred to the appropriate test medium. The medium used was Eagle's minimum essential medium (MEM)with Earle's salts and 0.23 mM pyruvate, supplemented with 3 mg/ml crystallized lyophilized bovine serum albumin (ICN Immunobiologicals, Lisle, IL). For microinjection experiments, denuded oocytes were cultured in 50-100 pl drops of medium covered with washed paraffin oil in a Falcon petri dish (#lo081 to minimize trauma after microinjection and to enable their direct observation. Cultures were carried out on a warming tray maintained at 37°C under a Plexiglas gassing cage

G-PROTEINS AND OOCYTE MATURATION

for 3 h. For longer incubations (17 h), petri dishes were transferred to modular incubator chambers and cultured in a water-jacketed incubator at 37°C. All other cultures were carried out in rubber stopper-covered borosilicate glass culture tubes immersed in a 37°C water bath. All media were gassed with a humidified mixture of 5% C02, 5% 02,and 90% N2.

Microinjection Denuded oocytes were isolated and washed in IBMX-supplemented medium and then transferred to a small drop of the same medium under washed paraffin oil. For some experiments, the medium contained 4 mM hypoxanthine instead of IBMX. All oocytes received 10 pl of the appropriate test compound or buffer control by microinjection, were washed four times through inhibitor-free medium, then transferred to a 100-pl drop of MEM/BSA under oil. Microinjection was carried out using pulled borosilicate capillaries mounted on Leitz mechanical micromanipulators adapted to an Olympus CK2 inverted microscope. Zona pellucida removal To address t h e possibility t h a t cholera toxin action on denuded oocytes is mediated by remnants of cumulus cells present within the zona pellucida, the zona pellucida was removed by either acid or pronase treatment, as described by Bornslaeger and Schultz (‘85),and the zona-less oocytes were exposed to the toxin. Cumulus cell-enclosed oocytes were isolated and then denuded in MEM containing 3 mgiml polyvinylpyrrolidone (PVP) and 0.2 mM IBMX prior to zona removal. For acid treatment, the denuded oocytes were placed in 154 mM NaC1, 10 mM H3P04, 3 mg/ml PVP, and 0.2 mM IBMX, pH 2.5 for 2-3 min. For pronase treatment, the oocytes were placed i n MEM/PVP containing 2 mgiml pronase and 0.2 mM IBMX for 10 min. After treatment, oocytes t h a t had no detectable zona pellucida were washed four times in MEM/BSA and transferred to the appropriate test medium.

393

medium was obtained from the last wash dish for each group and served as the blank. ?tYenty oocytecumulus cell complexes were assayed for the - FSH groups, whereas five complexes were assayed for the + FSH groups. cAMP was assayed using a commercial radioimmunoassay kit from DuPont. At the time of assay, samples were lyophilized and assayed according to the protocol provided. Each experiment was carried out six times with duplicate determinations per experiment.

Chemicals GDP, GTP, hypoxanthine, PVP, NaF, cholera toxin, pertussis toxin, and A1CI3 were obtained from Sigma Chemical Co. (St. Louis, MO). ATP,S, GDP$, GTPyS, and GppNHp were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Ovine FSH was generously provided by the Pituitary Hormone Distribution Program of the NIADDK. Concentrated stock solutions of the toxins were prepared by dissolving in PBS containing 3 mg/ml crystallized lyophilized BSA and were stored at 4°C for no longer than 1month. Aliquots of stock solutions were added to the medium to achieve the desired concentration. IBMX, purchased from Aldrich Chemical Co. (Milwaukee, WI), was first dissolved in 100% ethanol and the stock was diluted with medium. All other agents were added directly to culture medium.

Sta tis tics Germinal vesicle breakdown (GVB), the endpoint for oocyte maturation utilized herein, was scored using a Wild stereomicroscope. For the oocyte microinjection experiments, data were pooled and analyzed by chi-square analysis. All other oocyte maturation experiments were performed at least three times with a minimum of 50 oocytes assayed per group per experiment, and data were reported as mean percent SEM. The frequencies were subjected to arcsin transformation and were analyzed by Student’s t-test or by ANOVA followed by Duncan’s multiple range test. cAMP data were analyzed cAMP determination by ANOVA and Duncan’s multiple range test. SigCumulus cell-enclosed oocytes were isolated in nificant differences were inferred at P < 0.05. IBMX-containing medium, washed free of the inhibRESULTS itor, and transferred to a culture tube containing the appropriate test medium. After a 2-h incuba- Effecto fmicroinjected nucleotides and their analogs on the spontaneous maturation o f tion, cumulus cell-enclosed oocytes were washed denuded oocytes through four changes of medium containing 100 pM In initial experiments denuded germinal vesicleIBMX (to maintain cAMP levels), transferred t o stage mouse oocytes were microinjected with 10 pl glass tubes containing 0.1 ml ice cold 0.1 N HC1, of control buffer (PBS) or 10 pl of a 27 mM soluand frozen until assayed. An equal volume of

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Fig. 1. The action of nucleotides and nucleotide analogs on the spontaneous maturation of denuded oocytes. Denuded oocytes were microinjected with 10 pl of a 27 mM solution of each compound to produce a final intraoocyte concentration of 100 kM.Buffer-injected controls received the PBS vehicle. The oocytes were cultured 3 h in 0.5 ml drops MEM/BSA under oil on a warming tray. Statistical differences from control were analyzed by chi square (*, P < 0.05).

tion of one of a number of commercially available purine nucleotides or nucleotide analogs while meiotic arrest was maintained with IBMX, followed by a 3-h culture period in IBMX-free medium. The final intraoocyte concentration of each nucleotide was 100 pM. Microinjection of the PBS vehicle resulted in a frequency of maturation of 74% (Fig. 1).The majority of the nucleotides and analogs tested had no effect on oocyte maturation and these included GTP, GppNHp, GDP, GDP,S, and ATP,S. Only GTPyS reduced the frequency of maturation

below that of vehicle-injected controls, as only 26% GVB was observed in this group. The inhibitory effect of GTPyS was not likely due to a nonspecific effect of the sulfur on the terminal phosphate, because ATPyS, which has a similar structure, did not suppress GVB. Next, a sample of GTPyS purified by high performance liquid chromatography (HPLC)was used. This cleaner nucleotide preparation was the generous gift of Dr. Gregory Kopf at the University of Pennsylvania and controlled for the possibility that a contaminant in the less pure commercial preparation accounted for its inhibitory action on oocyte maturation. A different buffer, HEPES (10 mM, pH 7.2), was used as the nucleotide vehicle. Microinjection of this buffer into denuded oocytes resulted in a maturation frequency of 90%after 3 h of incubation. A dose response experiment utilizing concentrations of GTPyS that resulted in final intraoocyte concentrations from 1 to 50 pM produced a dose-dependent inhibition of GVB (Fig. 2). After assessing the frequency of GVB at 3 h, the denuded oocytes that were still arrested at the GV stage were returned to a drop of medium under oil, transferred to a modular incubator chamber and cultured overnight at 37°C. The denuded oocytes were assessed again for GVB after an additional 17 h of culture. As shown in Figure 2, the microinjected denuded oocytes were no longer maintained in meiotic arrest after the second culture period. These data demonstrate that the inhibitory action of GTPyS on spontaneous maturation of denuded oocytes is transient and, therefore, that its effect is not due to an irreversible toxic effect. 20 h

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Fig. 2. Dose response effect of GTPyS on spontaneous oocyte maturation of denuded oocytes. Denuded oocytes were microinjected with varying concentrations of GTPyS and cultured for 3 h in 0.5 ml drops of MEM/BSA under oil on a warming tray. The concentrations given are final intraoocyte concentrations (kM).After 3 h the oocytes were assayed for GVB and

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G-PROTEINS AND OOCYTE MATURATION

Further experiments were conducted to determine whether the inhibitory effect of GTPyS could be antagonized by GDPPS. It was reasoned that binding of the nonmetabolizable diphosphate analog to G-proteins would prevent binding of GTPyS and thus prevent activation (Gomperts, '83; Barrowman et al., '86). This approach has been used successfully in starfish oocytes to prevent l-methyladenine induction of maturation (Shille and Jaffe, '87). Denuded oocytes were microinjected with a mixture of GTP-yS and GDPPS that resulted in intraoocyte concentrations of 10 pM GTPyS and 25 FM or 100 pM GDPPS and cultured 3 h. The GDP analog had no beneficial effect at either concentration (data not presented). A second experiment involved a double microinjection protocol to determine whether initial exposure of the oocyte to GDPPS while maintained in meiotic arrest with IBMX would produce a reversal of the GTPyS effect. Oocytes were microinjected with either buffer or GDPPS (intraoocyte concentration 25 pM) and maintained in meiotic arrest 0.75 h with 0.2 mM IBMX. Those oocytes having survived the first microinjection were then microinjected a second time with GTPyS (intraoocyte concentration 10 pM) and cultured a n additional 3 h in IBMX-free medium. No difference was observed in the frequency of maturation between the two groups (bufferinjected 65% GVB, n = 20; GDPPS-injected 60% GVB,n = 15).

Effectsofmodifiers of G-protein activity on meiotic maturation We further examined the possible role of G-proteins in the control of mouse oocyte maturation by treating oocytes with chemical modulators of Gproteins under a variety of conditions. These modulators included: (1)cholera toxin, a n activator of the stimulator G-protein of adenylate cyclase, G,, (2) pertussis toxin, a n inactivator of the inhibitory G-protein of adenylate cyclase, Gi, as well as numerous other G-proteins, and (3) NaF plus A1CI3,hereafter referred to as AIF4-, a general stimulator of G-proteins. The first series of experiments focused on denuded oocytes and the possible involvement of oocyte G-proteins in the maintenance of meiotic arrest. The second series of experiments involved cumulus cell-enclosed oocytes. Cumulus cells are metabolically coupled to the oocyte in situ and directly influence the growth and development of the gamete. Therefore, they undoubtedly contain signal transduction systems involving G-proteins that are potential targets for the action of G-proteinmodifying agents.

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Fig. 3. The effects of modifiers of G-protein activity on meiotic arrest maintained with hypoxanthine in denuded oocytes. Cumulus cell-enclosed oocytes were isolated and denuded in medium containing 4 mM hypoxanthine and transferred to culture tubes containing the appropriate test medium for a 6 h culture period. Concentrations of the modifiers were: A1F4-, 20 mM NaF, 10 FM AlCl,; pertussis toxin (PT) and cholera toxin (CT), 100 FM. The - CT groups and the + CT groups were analyzed separately by ANOVA and Duncan's multiple range test. Bars within - CT or + CT groups with at least one identical letter at the top a r e not significantly different (P > 0.05).

Denuded oocytes

Hypoxanthine arrested. Denuded oocytes were cultured 6 h in medium containing 4 mM hypoxanthine plus varying combinations of the G-proteinmodifying drugs. When cultured in hypoxanthine alone, 56% of denuded oocytes underwent GVB (Fig. 3). Addition of cholera toxin to these cultures inhibited maturation (27%GVB), whereas supplementing hypoxanthine-containing medium with AlF4- promoted maturation (83%GVB). When cultures contained both A1F4- and cholera toxin, the frequency of maturation was identical to denuded oocytes treated with hypoxanthine alone. Thus the addition of AlF4- negated the inhibitory action of cholera toxin. Pertussis toxin had no effect on meiotic maturation in the presence or absence of cholera toxin. dbcAMP-arrest. The effects of these agents was next tested on the meiotic arrest maintained by dbcAMP, a meiotic inhibitor that more directly elevates intracellular CAMPlevels. Denuded oocytes were cultured 12 h in medium containing 300 pM dbcAMP and treated with each of the three agents. In the presence of dbcAMP alone, 16% of the oocytes underwent GVB (Fig. 4).Whereas neither pertussis toxin nor cholera toxin had a significant effect, AlF4- produced a modest increase in the frequency of GVB (35%). Effectofzonapellucida removal. The presence of functional adenylate cyclase within the oolemma

S.M. DOWNS ET AL.

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Fig. 4. Effects of G-protein modifiers on meiotic arrest maintained by dbcAMP in denuded oocytes. Denuded oocytes were cultured 12 h in medium containing 300 pM dbcAMP, k one of three G-protein modifiers (concentrations as in legend to Fig. 3).Statistical differences from control were determined by Student's t-test (*, P < 0.05).

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Fig. 5. The effect of zona pellucida removal on the inhibitory action of CT on denuded oocytes. In the hypoxanthine group, the zona pellucida was removed from oocytes by acid treatment and the resulting zona-free denuded oocytes were cultured 6 h in 4 mM hypoxanthine, t CT. In the IBMX group, the zona pellucida was removed from oocytes by pronase treatment and the resulting zona-free denuded oocytes were cultured 6 h in 50 pM IBMX, -+ CT. An asterisk at the top of the bar denotes a significant difference (P< 0.05)from the corresponding - CT group, by Student's t-test.

has been a subject of some controversy. The consistent inhibitory action of cholera toxin on denuded oocyte maturation suggests that activation of this enzyme in the oocyte by the bacterial toxin (through ADP ribosylation of the a subunit of G,) stimulates an increase in intracellular CAMP,which leads failed to produce inhibition of maturation (88% to a n inhibition of GVB. However, the denuded GVB). It was possible t h a t pronase treatment oocytes used in these experiments still had an intact digested a surface protein that played a n important zona pellucida containing remnants of the cumu- role in the inhibitory action of hypoxanthine. Therelus cells (Eppig and Downs, '84; Bornslaeger and fore, a similar experiment was performed in which Schultz, '85; Canipari e t al., '88). It was therefore pronase-treated oocytes were cultured 6 h in 50 pM possible that the action of cholera toxin on the IBMX, in the presence or absence of cholera toxin. oocyte was mediated by the cumulus cell processes With the zona pellucida intact, few denuded oocytes embedded in the zona pellucida. To test this possi- underwent GVB in this concentration of IBMX bility, the zona pellucida was removed before chol- (12%),and cholera toxin had little further effect era toxin treatment by brief exposure to either acid on maturation (8%GVB; Fig. 5 ) . However, whereas or pronase treatment. Both of these treatments have pronase treatment resulted in an increased matubeen shown to reduce the number of cumulus cell ration frequency of 52% in IBMX-treated oocytes, remnants associated with the oocyte (Bornslaeger the addition of cholera toxin still produced a decrease in the frequency of GVB to 25%. and Schultz, '85). When the zona pellucida was removed from Effect ofpertussis toxinpretreatment. Treatment denuded oocytes by acid treatment and zona-free of denuded oocytes with pertussis toxin had little oocytes were incubated 6 h in 4 mM hypoxanthine, effect on meiotic maturation, and it is possible that no difference in the frequency of maturation was the lack of effect of this bacterial toxin is due to observed when compared to oocytes with intact the need for a n extended period of incubation before zonae pellucidae (47%vs. 50%GVB; Fig. 5).In addi- its action is manifested. Denuded oocytes were tion, removal of the zona pellucida with acid did therefore first incubated 4 h in medium containnot prevent the inhibitory action of cholera toxin ing IBMX pertussis toxin to allow time for peron oocyte maturation (30%GVB with zona pellu- tussis toxin action to be manifested while oocytes cida, 33%GVB without). When the zona pellucida were maintained in meiotic arrest. The oocytes were was removed by pronase, a dramatic increase in the then washed free of inhibitor and cultured a subfrequency of maturation in control cultures oc- sequent 4-h period in hypoxanthine-containing curred, from 43%to 92% GVB. Furthermore, treat- medium ? cholera toxin. Cholera toxin was used ment of the zona-free oocytes with cholera toxin in the second incubation because of its demon_+

G-PROTEINS AND OOCYTE MATURATION

strated inhibitory action on the denuded oocyte. Only those cultures exposed to pertussis toxin in the initial incubation were treated with the toxin in the second incubation. Consistent with the results in Figure 3, cholera toxin suppressed maturation in hypoxanthine-containing medium (30.4% compared t o 56.1% GVB in cholera toxin-free medium). The addition of pertussis toxin to hypoxanthine-containing medium had no significant effect on maturation whether cholera toxin was present or absent (26.3% and 53.5% GVB, respectively). Cumulus cell-enclosedoocytes Hypoxanthine-arrested. Cumulus cell-enclosed oocytes were maintained in meiotic arrest for 12 h with 4 mM hypoxanthine. At this concentration, 41%of the cumulus cell-enclosed oocytes underwent GVB (Fig. 6). Addition of A1F4- resulted in a dramatic increase in maturation frequency to 94%.Pertussis toxin (100 pM) failed to produce a n effect. However, cholera toxin (100 pM) increased the frequency of GVB to 63%.In addition, oocytes from the cholera toxin-treated groups exhibited varying degrees of blebbing in the perivitelline space. FSH-arrested. FSH has been shown to arrest transiently the spontaneous maturation of cumulus cell-enclosed oocytes during short-term culture (Eppig et al., '83). Therefore, the effect of modifiers of G-proteins on this inhibitory process was tested. Cumulus cell-enclosed oocytes were cultured 2 h in the presence or absence of FSH and to these cultures was added cholera toxin, pertussis toxin 100

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Fig. 6. The effects of modifiers of G-protein activity on meiotic arrest maintained by hypoxanthine in cumulus cell-enclosed oocytes. Oocyte-cumulus cell complexes were isolated in medium containing 4 mM hypoxanthine and transferred to culture tubes containing the appropriate test medium (concentrations of the modifiers as in legend to Fig. 3). Statistical differences from control were analyzed by Student's t-test (*, P < 0.05).

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Fig. 7. The effects of G-protein modifiers on FSH-maintained meiotic arrest (A) and FSH-stimulated CAMP formation (B) in cumulus cell-enclosed oocytes. A: Oocyte-cumulus cell complexes were cultured 2 h in medium containing or lacking G-protein modifier, 2 0.1 pg/ml FSH (concentrations of the modifiers as in legend to Fig. 3). B: Oocyte-cumulus cell complexes were isolated and cultured as described above. For both A and B, the - FSH and + FSH groups were analyzed separately by ANOVA and Duncan's multiple range test. Bars within -FSH o r + FSH groups with a t least one identical letter a t the top are not significantly different ( P > 0.05). In B, differences between - FSH and + FSH in each treatment group were also analyzed by Student's t-test and, in each instance, the difference was significantly different ( P < 0.05).

or AlF4-. In FSH-free control medium, 97% of the cumulus cell-enclosed oocytes underwent maturation (Fig. 7A). FSH reduced this percentage to 69%. The addition of AlF4- to FSH-free medium produced only a slight drop in GVB (to 93%),but this agent reduced the inhibitory action of FSH on oocyte maturation (87%GVB). Pertussis toxin also produced a slight drop in GVB (to 94%) in FSHfree medium but did not alter FSH-maintained meiotic arrest. Cholera toxin alone reduced the frequency of GVB to 72% and augmented the inhibitory action of FSH in an additive manner (45%GVB).

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398 100 V

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Fig. 8. The effects of G-protein modifiers on meiotic arrest and FSH-induced maturation in dbcAMP-arrested, cumulus cell-enclosed oocytes. Cumulus cell-enclosed oocytes were cultured 12 h in medium containing 300 )LMdbcAMP plus one of three G-protein modifiers, ? 0.1 pg/ml FSH (concentrations of modifiers as in legend to Fig. 3). The - FSH and + FSH groups were analyzed separately by ANOVA and Duncan’s multiple range test. Bars with identical letters a t the top are not significantly different (P> 0.05). As determined by Student’st-test, a significant increase in GVB was stimulated by FSH in only the control and PT-treated groups.

A second set of oocyte-cumulus cell complexes, treated in identical fashion, was assayed for cAMP levels after 2 h incubation. Complexes cultured in MEM/BSA contained about 2 fmol cAMP per complex (Fig. 7B). FSH increased this value over 13-fold to 26 fmol. AlF4- had no effect on basal cAMP levels; however, of particular interest was the finding that AlF4- increased cAMP levels in the presence of FSH above the level obtained with FSH alone (42 fmol/complex). Pertussis toxin had no effect on cAMP levels, either in the presence or absence of FSH. The results with cholera toxin were consistent with the GVB assay data. Cholera toxin alone elevated cAMP levels (17 fmol/complex),and, like its interaction with FSH on oocyte maturation, the effect of cholera toxin and FSH on cAMP was additive (41 fmol/complex). dbcAMP-arrested. The actions of A1F4~,pertussis toxin, and cholera toxin were subsequently tested on the meiotic arrest maintained by dbcAMP in cumulus cell-enclosed oocytes. Cumulus cell-enclosed oocytes were cultured 12 h in medium containing300 pM dbcAMPand treated with one of the three agents. In the presence of dbcAMP alone, only 15%GVB was observed (Fig. 8).The addition of A1F4- or pertussis toxin had no effect on maturation, but cholera toxin increased the incidence of GVB to 79%.

FSH-stimulated. Because AlF4- did not stimulate maturation in dbcAMP-arrested cumulus cellenclosed oocytes, it provided a paradigm t o test the action of this agent, as well as cholera toxin and pertussis toxin, on FSH-stimulated maturation that occurs during longer term cultures. When FSH was added to dbcAMP-containingmedium, oocyte maturation was increased by 67% after 12 h (82%GVB; Fig. 8). When FSH-containing medium was supplemented with A1F4-, this positive response to gonadotropin was completely suppressed. Pertussis toxin and cholera toxin were without effect in FSH-treated cultures. It is important to note that cholera toxin did not act synergistically with FSH to increase further the frequency of maturation. Effect of pertussis toxin pretreatment. Cumulus cell-enclosed oocytes were subjected to a pertussis toxin pretreatment protocol to test whether increasing the exposure time to the toxin while the oocyte was maintained in meiotic arrest would result in a significant inhibitory response. Cumulus cellenclosed oocytes were isolated and cultured 2 h in medium containing 200 pM IBMX plus or minus 100 pM pertussis toxin. At the end of this initial culture period, the cumulus cell-enclosed oocytes were washed through four changes of IBMX-free medium and subsequently incubated for 1.5 h in the presence or absence of FSH. Those complexes that had been incubated initially in medium containing pertussis toxin were also exposed to pertussis toxin during the second incubation. FSH reduced the incidence of maturation from 90% t o 51%. Pertussis toxin treatment had no effect on the maturation of oocytes in FSH-free medium (89% GVB); however, the toxin reduced the frequency of FSHtreated cultures (19%GVB; P < 0.05).

DISCUSSION The present study provides evidence that G-proteins participate in the mechanisms governing meiotic maturation in mouse oocytes. The inhibitory action of microinjected GTPyS on spontaneous GVB in denuded oocytes is consistent with the activation of a G-protein with resultant production of a mediator that suppresses maturation. The possibility that oocyte adenylate cyclase is stimulated, leading to generation of meiosis-arresting levels of CAMP,is supported by the finding that cholera toxin suppresses GVB in denuded oocytes and cumulus cell-enclosed oocytes during short-term culture, and this was associated with elevation of cAMP levels. Cholera toxin also stimulated the resumption of maturation during 12 h culture when meiotically arrested oocytes were enclosed by an intact cumu-

G-PROTEINS AND OOCYTE MATURATION

lus oophorus. Pertussis toxin had little effect on the maintenance of meiotic arrest or hormone-induced maturation, except that treatment of oocyte-cumulus cell complexes produced a n augmentation of the inhibitory action of FSH. A1F4- produced disparate effects consistent with a n antagonistic action on cAMP action. This agent stimulated maturation when meiotic arrest was maintained with hypoxanthine or dbcAMP and prevented the transient arrest in cumulus cell-enclosed oocytes produced by FSH, yet FSH-induced resumption of maturation was suppressed. These data support the participation of G-proteins at both the oocyte and cumulus cell levels in the control of mouse oocyte maturation.

Action o f GTP analogs When oocytes were microinjected with nucleotides, only GTPyS produced inhibition of meiotic maturation. The specificity of the GTPyS effect as well as its transient arresting action argues against a nonspecific toxicity. The results are consistent with the activation of G-protein by GTP, association with the appropriate effector molecule, and subsequent production of a second messenger(s) such as cAMP or a phosphoinositide metabolite that mediates meiotic arrest. The lack of effect with microinjected GTP could be explained by a rapid hydrolysis of the terminal phosphate when compared to the nonmetabolizable analog. However, Gpp(NH)p, another analog resistant to hydrolysis, also failed to elicit a n inhibitory effect. This is not particularly surprising, considering a precedent in the literature for a greater potency with the GTPyS analog (e.g., Helpler and Harden, '86; Pfeilschifter and Bauer, '87; Stutchfield and Cockcroft, '88; Regazzi et al., '89). The difference in potency could be due to differential affinity of the G-proteids) for the nucleotide analogs. The lack of effect of microinjected GDPPS on GTPyS-maintained meiotic arrest indicates that the diphosphate analog competes poorly with exogenous GTPyS at the concentrations tested. Action of cholera and pertussis toxins Cholera toxin and pertussis toxin, bacterial toxins that catalyze the ADP-ribosylation of the (Y subunit of G-proteins, were utilized in the present study to investigate further the interaction of G-proteins with the control of oocyte maturation. G,, the stimulatory G-protein for adenylate cyclase, and Gt, the retinal transducin G-protein, are the targets of cholera toxin action, and covalent modification of dissociated a subunit by the toxin attenuates GTPase activity; in the case of G,, this

399

constitutively activates adenylate cyclase (Cassel and pfeuffer, '78; Moss and Vaughan, '79). Pertussis toxin, in contrast, has a greater number of possible substrates, including Gi, the inhibitory Gprotein for adenylate cyclase (Katada, '82), Gt (Woodruff and Bownds, '791, Go from brain (Sternweis and Robishaw, '84;Neer et al., '841, G,, which stimulates phospholipase C (Cockcroft, '87), and &, involved in gating of ion channels (Brown and Birnbaumer, '88). Covalent modification of heterotrimeric G-protein by pertussis toxin prevents dissociation of py subunits from cx and thus suppresses activation of the protein (Katada et al., '84; Bokoch et al., '84). The actions of cholera toxin on denuded oocytes and cumulus cell-enclosed oocytes are consistent with activation of G, and elevated levels of CAMP. Increased cAMP in the oocyte is correlated with maintenance of meiotic arrest (Schultz et al., '83), and adenylate cyclase has been identified on the plasma membrane of bovine oocytes by cytochemical localization (Kuyt et al., '88). In addition, when denuded oocytes are treated with forskolin, a potent activator of adenylate cyclase, meiotic arrest is maintained in vitro, in conjunction with stimulation of cAMP synthesis (Olsiewski and Beers, '83; Schultz et al., '83; Urner and Schorderet-Slatkine, '84).A demonstrated action of cholera toxin on the denuded oocyte is more elusive, however. The spontaneous maturation of denuded oocytes in inhibitorfree medium is not affected by exposure to cholera toxin, which is consistent with a lack of effect on oocyte cAMP (Olsiewski and Beers, '83; Schultz et al., '83). Nevertheless, if a partial arrest of meiotic maturation is maintained by CAMP-elevating agents, a n inhibitory action of the toxin can be demonstrated (Vivarelli et al., '83; Downs and Eppig, '85; data herein). Similarly, elevation of oocyte cAMP by cholera toxin has been achieved, but only when aided by the inclusion of FSH or IBMX (Vivarelli et al., '83; Olsiewski and Beers, '83).Thus cholera toxin alone probably generates a limited amount of cAMP that is unable to maintain meiotic arrest due to rapid hydrolysis by phosphodiesterase; the addition of phosphodiesterase inhibitors or CAMP-elevating agents likely raises the level of cAMP above a n inhibitory threshold, resulting in suppression of meiotic maturation. The small inhibitory action of cholera toxin on GVB in denuded oocytes is not likely due to a limiting concentration of the toxin, because a 20-fold higher dose exhibited a similar potency in dbcAMP-arrested denuded oocytes (Downs and Eppig, '85). The results of cholera toxin on cumulus cell-

400

S.M. DOWNS ET AL.

enclosed oocytes provide strong support for the mediation of both meiotic arrest and induction of maturation by CAMP. During 2 h cultures, the effects of cholera toxin and FSH on both the maintenance of meiotic arrest and in the generation of cAMP in the complexes were additive. In addition, cholera toxin stimulated maturation in cumulus cell-enclosed oocytes during 12 h cultures when meiotic arrest was maintained with either hypoxanthine or dbcAMP. The toxin did not elicit maturation in denuded oocytes, demonstrating an important role of the cumulus cells in mediating the stimulatory response. Therefore, CAMP,likely generated in the cumulus cells in response to cholera toxin interaction with G, and adenylate cyclase, is inhibitory to GVB during short-term cultures but stimulates the resumption of maturation in longterm cultures. This agrees well with previous data utilizing FSH (Downs et al., '88; Downs, '90a). Pertussis toxin has been shown t o stimulate the ADP-ribosylation of a Gi-like protein in mouse oocytes (Allworth et al., '90; Jones and Schultz, '90). In addition, Jones and Schultz ('90) report that, when pretreated with pertussis toxin, the spontaneous maturation of denuded oocytes was slightly accelerated.We were unable to demonstrate any significant action of the toxin on dbcAMP-, hypoxanthine-, or cholera toxin-treated denuded oocytes. Even when denuded oocytes were pretreated for 4 h while maintained in meiotic arrest, pertussis toxin failed to exert an effect. These data show that, despite the presence of ADP-ribosylatable substrate, pertussis toxin failed to exert a direct influence on the maturation of denuded oocytes. However, it is likely that differences in experimental conditions are responsible for the disparate results in these two studies. Jones and Schultz examined effects of the toxin on spontaneous maturation during shortterm culture, whereas we tested its effects on meiotically arrested oocytes during longer incubation times. Pertussis toxin also had a limited impact on the maturation of cumulus cell-enclosed oocytes in the present study. The one significant effect of the toxin was on FSH inhibition of maturation in cumulus cell-enclosedoocytes during short-term culture, but this occurred only after a 2-hour pretreatment period in PT-containing medium (the frequency of GVB was reduced from 51%to 19%).These results suggest an effect on Gi, the G-protein that exerts an inhibitory control over adenylate cyclase, because its inactivation by pertussis toxin would allow a greater response of the enzyme to FSH and lead to higher cAMP levels and greater suppression of GVB during short-term culture. Although no effect of PT

on cAMP levels was observed when PT was added concurrently with FSH, it is possible that pretreatment of complexes with pertussis toxin resulted in increased cAMP levels within the oocyte-cumulus cell complex upon subsequent treatment with gonadotropin. Alternatively, pertussis toxin treatment could result in lowered levels of Py dimer (cf, Watkins et al., '89), which would favor the dissociated, activated state of Gsa, or perhaps ADP-ribosylation of another G-protein involved with a different effector molecule.

Action of AIF, Fluoride has been used for many years as a general stimulator of G-proteins. This halogen requires aluminum for its activity (Sternweis and Gilman, '€32)' and the complex,A1F4-, stimulates G-proteins by mimicking the terminal phosphate of GTP (Bigayet al., '87). AlF4- stimulates many different G-proteins, including G, (Gilman, '87), Gi (Katada et al., '84),Gt (Kanaho et al., '851, and G, (Blackmore et al., '85; Strnad and Wong, '85). AlF4- had a direct, maturation-promoting action on the oocyte. When denuded oocytes were maintained in meiotic arrest with hypoxanthine or dbcAMP, this agent produced an increase in the frequency of GVB; AlF4- also prevented the inhibitory action of cholera toxin on maturation of denuded oocytes. Jones and Schultz ('90) proposed that the slight acceleration of spontaneous maturation in response to pertussis toxin might be due to inactivation of the G-protein involved in the activation of phospholipase C; this would prevent the stimulation of protein kinase C, which has been shown to suppress spontaneous maturation of GVB (Urner et al., '83;Bornslaeger et al., '86)and thereby accelerate the kinetics of GVB. If protein kinase C mediates the action of the activated G, on oocyte maturation, then A1F4- is probably not acting through this protein, since activation would be expected t o produce the opposite effect of that observed-i.e., maintenance of meiotic arrest. It is, of course, possible that another product of phospholipase C activity, inositol triphosphate (IP3),could modulate the meiotic state through fluxes in internal calcium concentration. However, microinjection of IP3failed to induce maturation in hypoxanthineor IBMX-arrested oocytes (Buccione and Eppig, unpublished results). This result, of course, does not rule out the possible involvement of IPSor other intermediates of the phosphoinositide pathway in the control of meiotic arrest. Indeed, Homa et al. ('91) demonstrated that microinjection of IPSinto IBMX-arrested bovine oocytes promoted meiotic

G-PROTEINS AND OOCYTE MATURATION

maturation. Additionally, an unknown G-protein could be the target for AlF4-. Both inhibitory and stimulatory actions on oocyte maturation were observed in cumulus cell-enclosed oocytes in response to A1F4-, but these seemingly contradictory actions may be reconciled if one considers them as antagonistic to the action of CAMP. The actions of FSH are generally thought to be mediated by CAMP.AlF4- increased the level of cAMP in FSH-stimulated oocyte-cumuluscell complexes, perhaps through interaction with G,, yet suppressed both inhibitory and stimulatory effects of this gonadotropin on oocyte maturation. This suggests pleiotropic effects on the mechanisms influencing oocyte maturation and could involve different G-proteins. The mode of hypoxanthine action on oocyte maturation is similarly through control of cAMP levels (Downs et al., '89), and AlF4reversed the inhibitory effect of this purine on oocyte maturation. It is therefore likely that the maturation promoting actions of AlF4- are manifested downstream of cAMP production, either preventing the activation of protein kinase A or modifying substrate phosphorylation states at a distal point (see below). The one exception to this scheme is the response of oocytes in the presence of dbcAMP. Although A1F4- stimulated an increase (albeit limited) in GVB in dbcAMP-arrested denuded oocytes, it had no effect in similarly treated cumulus cellenclosed oocytes. The significance of this observation is not readily apparent but may represent a unique feature of this particular cAMP analog. AlF4- behaves like the terminal phosphate in GTP-bound, activated G-proteins, but its very na-

401

ture as a phosphate analog also makes it a prime candidate for inhibition of phosphatases (Bigay et al., '87). Thus the actions ofA1F4- could be exerted through the suppression of phosphatase activity. Support for this idea comes from the finding that microinjection of phosphatases or phosphatase inhibitors into starfish or frog oocytes modulates meiotic maturation (Huchon et al., '81; Foulkes and Maller, '82; Hermann et al., '84;Meijer et al., '86; Pondaven and Meijer, '86). In addition, the protein phosphatase 1- and 2A-specific inhibitor, okadaic acid (Cohen et al., ,901, has been shown to stimulate a burst of phosphorylation and generation of maturation promoting factor in frog and starfish oocytes (Goris et al., '89; Pondaven et al., '89; Picard et al., '89; Rime et al., '901, and, most recently, to stimulate maturation of mouse oocytes maintained in meiotic arrest by a variety of arresting substances (Rime and Ozon, '90; Alexandre et al., '91; Gavin et al., '91; Schwartz and Schultz, '91). Thus, A1F4- could interfere with phosphorylationidephosphorylation reactions involved in controlling the activation state of MPF (Hunt, '89; Lewin, '90) and influence the G2/M cell cycle transition point characterizing the germinal vesicle stage. This effect might not necessarily result from direct modulation of G-protein activity. A1F4- could also affect the phosphorylation state of G-proteins themselves and thereby alter their activity (Carlson et al., '89; Pyne et al., '89).

Conel usion Table 1summarizes the actions of G-protein modifiers on maturation of mouse oocytes in vitro. It is

TABLE 1 . Summary of actions of G protein modifiers on mouse oocyte maturation Treatment I. HX- or dbcAMParrested oocytes + CT

+ PT + AlF4-

CEO or DO

Inhibitory ( - ), Maturation-Promoting or No Effect (NE) on maturation

DO CEO DO CEO DO CEO

-

NE NE

CEO CEO CEO

NE (- when pretreated)

CEO CEO CEO

NE NE

+

+ (HX-arrested), NE (dbcAMP-arrested) -

+

111. FSH-stimulated (17-18h cultures)

+ CT +PT + AlF4

+)

+

11. FSH-arrested (2 h cultures)

+ CT + PT + AlF4-

(

-

S.M. DOWNS ET AL.

402

evident from these data as well as the results of the microinjection experiments that a multitude of heterotrimeric G-proteins could be involved in the regulation of oocyte maturation. In addition, GTP-binding proteins of the small type could also participate in these signal transducing interactions (Burgoyne, '89; Hall, '90). The promiscuity of pertussis toxin and A W - interactions with different G-proteins as well as the interactions of the Pr dimer with different a subunits underscores the need for caution in interpreting these results. Further potential difficulty might be encountered with differential effects of G-proteins in the oocyte and in the follicle cells that regulate the meiotic state of the oocyte. Whereas the present study implicates G-proteins in the control of germinal vesicle breakdown in mouse oocytes, more work will be required before specific physiologicalfunctions can be defined.

ACKNOWLEDGMENTS Thanks to Mark Dow and Philip Caron for dedicated technical assistance. Thanks also to Dr. Peter Hoppe for the use of his lab facilities for producing microinjection pipets. This study was supported by funds from the NIH (HD25291 to S.M.D. and HD20575 to J.J.E.). The Jackson Laboratory is fully accredited by the American Association for the accreditation of Laboratory Animal Care. LITERATURE CITED Alexandre, H., A. Van Cauwenberge, Y. Tsukitani, and J. Mulnard (1991) Pleiotropic effect of okadaic acid on maturing mouse oocytes. Development, 112:971-980. Allworth, A.E., J.D. Hildebrandt, and C.A. Ziomek (1990) Changes in the amount of a pertussis toxin ribosylatable G-protein with egg maturation and early development in the mouse. Dev. Biol., 142:129-137. Bahnson, T.D., S.-C. Tsai, R. Adamik, J . Moss, and M. Vaughan (1989) Microinjection of a 19-kDguanine nucleotide-binding protein inhibits maturation ofXempus oocytes.J. Biol. Chem., 264:14824-14828. Bigay, J., P. Deterre, C. Pfister, and M. Chabre (1987) Fluoride complexes of aluminum or beryllium act on G proteins as reversibly bound analogues of the y phosphate of GTl? EMBO J.,6:2907-2913. Birchmeier, C., D. Broek, and M. Wigler (1985) ras proteins can induce meiosis inXenopus oocytes. Cell., 43:615-621. Birnbaumer, L., J . Codina, R. Mattera, R.A. Cerione, J.D. Hildebrandt, T. Sunyer, F.J. Rojas, M.G. Caron, R.J. Lefkowitz, and R. Iyengar (1985) Regulation of hormone receptors and adenylyl cyclases by guanine nucleotide binding proteins. Rec. Prog. Horm. Res., 41 :41-94. Blackmore, P.F., S.B. Bocckino, L.E. Waynick, and J.H. Exton (1985) Role of a guanine nucleotide-binding regulatory protein in the hydrolysis of hepatocyte phosphatidylinositol 4,5-biphosphate by calcium-mobilizing hormones and the control of cell calcium. Studies utilizing aluminum fluoride. J . Biol. Chem., 260:14477-14483. Bokoch, G.M., T. Katada, J.K. Northum, M. Ui, and A.G. Gil-

man (1984) Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J. Biol. Chem., 259:3560-3567. Bornslaeger, E.A., W.T. Poueymirou, P.M. Mattei, and R.M. Schultz (1986) Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes. Exp. Cell Res., 165507-517. Bornslaeger, E.A., and R.M. Schultz (1985) Adenylate cyclase activityinzona-freemouseoocytes. Exp. CellRes., 156.277-281. Brown, A.M., and L. Birnbaumer (1988) Direct G protein gating of ion channels. Amer. J . Physiol., 254:H401-H410. Burgoyne, R.D. (1989) Small GTP-binding proteins. TIBS, 14:394-396. Canipari, R., A. Bevilacqua, R. Colonna, M. De Felici, and F. Mangia (1988) Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes. Gamete Res., 20:115-124. Carlson, K.E., L.F. Brass, and D.R. Manning (1989) Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. J . Biol. Chem., 264:13298-13305. Cassel, D., and T. Pfeuffer (1978) Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc. Natl. Acad. Sci. USA, 7526694673. Cho, W.K., S. Stern, and J.D. Biggers (1974) Inhibitory effect of dibutyryl cAMP on mouse maturation in vitro. J. Exp. Zool., 187:383-386. Cockcroft, S. (1987) Polyphosphoinositide phosphodiesterase: Regulation by a novel guanine nucleotide binding protein, G,. TIBS, 12:75-78. Cohen, l?, F.B.C. Holmes, and Y. Tsukitani (1990) Okadaic acid: a new probe for the study of cellular regulation. TIBS, 5:98-102. Deshpande, A.K., and H.-F. Kung (1987) Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 MS proteins. Mol. Cell Biol., 7:1285-1288. Downs, S.M. (1990a) Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes. Molec. Reprod. Dev., 27.235-243. Downs, S.M. (1990b)The maintenance of meiotic arrest in mammalian oocytes. In: Fertilization in Mammals. Bavister, B., ed. Serono Symposia, USA, Norwell, MA, pp. 5-16. Downs, S.M., D.L. Coleman, and J.J. Eppig (1986) Maintenance of murine oocyte meiotic arrest: Uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes. Dev. Biol., 117:174-183. Downs, S.M., D.L. Coleman, P.F. Ward-Bailey, and J.J. Eppig (1985) Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc. Natl. Acad. Sci. USA, 82:454-458. Downs, S.M., S.A.J. Daniel, E.A. Bornslaeger, P.C. Hoppe, and J.J. Eppig (1989) Maintenance of meiotic arrest in mouse oocytes by purines: Modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Res., 23:323-334. Downs, S.M., S.A.J. Daniel, and J.J. Eppig(1988) Induction of maturation in cumulus cell-enclosed mouse oocytes by folliclestimulating hormone and epidermal growth factor: Evidence for a positive stimulus of somatic cell origin. J . Exp. Zool., 245:86-96. Downs, S.M., and J.J. Eppig (1985) A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate-dependent meiotic arrest in murine oocytes. Gamete Res., 11:83-97. Downs, S.M., and J.J. Eppig (1987) Induction of mouse oocyte

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