Role of progesterone on oocyte maturation in the egg-brooding hylid frog Gastrotheca riobambae (Fowler)

In the egg-brooding frog Gastrotheca riobambae (Fowler), oocyte maturation is comparable to the situation of other frog species. In isolated follicles, progesterone induces only germinal vesicle breakdown (GVBD), while human chorionic gonadotropin (hCG) induces GVBD and ovulation. In addition, defolliculated oocytes respond with GVBD to the treatment with progesterone, while hCG has no effect. As in other frogs, oocyte maturation in vitro depends on hormonal action and on the presence of divalent cations. In this frog, progesterone or a similar hormone conditions the brooding pouch for reproduction and induces pouch closure. Follicles from frogs with closed pouches showed GVBD after 15-17 hours of incubation with progesterone, while those from frogs with open pouches took 19-24 hours for GVBD. These findings suggest that follicles become stimulated for maturation when the pouch is closed and that this stimulated condition is maintained for several weeks in advance of the process of oocyte maturation. In G. riobambae, the external appearance of the pouch aperture indicates the reproductive condition of the ovary.     

Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.     

Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH

The effect of the different follicular cell types on resumption of meiosis was studied during stimulation with FSH. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), and cumulus and mural granulosa cells were used. The resumption of meiosis and oocyte maturation were assessed by the determination of the germinal vesicle breakdown (GVBD) and polar body formation (PB) at the end of a 24 hr culture period in the presence of 4 mM hypoxanthine (HX). The effects of recombinant LH (r-LH) and hCG were also evaluated. Oocyte exposure to the gonadotrophins varied from 5 min to 24 hr (i.e., priming time). Oocytes were obtained from immature gonadotrophin-stimulated and -unstimulated mice. 1. FSH (1 IU/L-75 IU/L) provoked a dose-dependent increase in GVBD and PB in CEO, but not in DO, in stimulated and unstimulated mice. Eight IU/L was sufficient for inducing resumption of meiosis. In contrast, LH and hCG (both 1 IU/L-1500 IU/L) were without effect on GVBD and PB in CEO and DO of oocytes from stimulated and unstimulated mice. A combination of 8IU/L FSH and 4–8 IU/L hCG produced an additive effect, whereas combinations with LH and higher concentrations of hCG had no such effect. 2. A 2 hr priming with FSH (8 IU/L-75 IU/L) induced a dose-dependent oocyte maturation in CEO. Thirty minutes of priming with FSH (75 IU/L) was sufficient for induction of meiotic resumption in CEO. 3. Priming CEO with FSH for 2 hr followed by the separation and repooling of oocytes and cumulus cells induced oocyte maturation. GVBD of new, unprimed DO added to cumulus cells of primed CEO increased slightly but was significant, whereas GVBD in DO isolated from the primed CEO only increased marginally. DO cocultured with FSH-primed cumulus masses seem to be prevented from resuming meiosis. 4. Priming a coculture of granulosa cells and DO with FSH for 2 hr caused a significant increase in GVBD compared to the control, evaluated after 24 hr. In contrast, a 24 hr FSH-priming of a coculture of granulosa cells and DO was without effect on GVBD. 5. A spent medium in which unstimulated cumulus cells or mural granulosa cells had grown was without effect on GVBD in DO. However, a small fraction of the DO resumed meiosis after culture in a spent medium derived from a 2 hr priming of CEO and spent media from 24 hr priming of CEO induced a 2–3 times higher GVBD frequency in the DO compared to the controls. Heat treatment of spent media (70°C, 30 min) from a 24 hr FSH-priming of CEO still induced GVBD in naive DO. The results showed that FSH, in a concentration of as little as 8 IU/L, but not r-LH and hCG, induced within 30 minutes the cumulus cells to produce and after 2 hr to secrete a diffusible heat stable meiosis activating substance. This substance overcame, in a paracrine fashion, the inhibiting effect of HX and induced oocyte maturation directly in DO. The production of this substance, however, was dependent on the initial connection between the cumulus cells and the oocyte, indicating an important 2-way communication between these 2 cell types. The mural granulosa cells did not produce a meiosis inducing activity by stimulation with FSH, but significantly, more DO matured after coculture with the nonstimulated granulosa cells for 24 hr than for 2 hr. It is proposed that the heat stable meiosis activating component of the spent media from the FSH-stimulated CEO belongs to the meiosis activating sterols, MAS, previously isolated from human follicular fluid and from adult bull testes. Mol. Reprod. Dev. 46:296–305, 1997. © 1997 Wiley-Liss, Inc.     

Internal pH of Xenopus oocytes: a study of the mechanism and role of pH changes during meiotic maturation

The internal pH (pHi) of Xenopus laevis oocytes, as measured by the DMO method, covered a broad range of values from 7.06 +/- 0.01 to 7.93 +/- 0.01, with a mean value of 7.43 +/- 0.03. The pHi measured by DMO and microelectrodes was nearly identical in control and maturing oocytes from the same batch. The oocytes from most females elevated their pHi in response to progesterone, reaching a maximum elevation of 0.30 +/- 0.03 pH units above control values at 100% germinal vesicle breakdown (GVBD). However, some females were found to contain oocytes that already had an elevated pHi of 7.71 +/- 0.03 which did not significantly increase during maturation. Human chorionic gonadotrophin (hcG)-stimulated females had oocytes with slightly higher control pHi values than oocytes from nonstimulated females but still showed the same elevation in response to progesterone. Thus, the "stimulated" state of oocyte physiology as induced by hcG did not account for the variation in control pHi and responsiveness to progesterone. Other aspects of this variability are discussed. Elevating or lowering the external pH is shown to elevate and lower pHi, respectively, in a stable and predictable manner. Using this approach to change pHi we have found no effect of changes in pHi on the rate of protein synthesis in control and maturing oocytes. Similarly, pHi had only a slight facilitating effect on the rate of GVBD. A pH indicator gel was used to demonstrate that the pHi increase during oocyte maturation involved an acid efflux. We conclude that an elevated pHi is not necessary for oocyte maturation, yet the mechanism of the pHi elevation is discussed as a possible lead to events that are necessary.     

Cyclic AMP-mediated control of oocyte maturation in the catfish, Clarias batrachus (Bloch): effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and phosphodiesterase inhibitors

An increase in the percentage of germinal vesicle breakdown (GVBD) with a corresponding decrease in cAMP was found in the oocytes which were incubated for 36 hr with different concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP). At its highest concentration (1 microgram/ml), 17 alpha,20 beta-DP induced 91.9 +/- 2.3% GVBD and decreased cAMP level to 0.8 +/- 0.1 pmol/oocyte from 2.9 +/- 0.2 pmol/oocyte (control). The two different known inhibitors of phosphodiesterase viz. 3-isobutyl-1-methyl-xanthine (IBMX) and theophylline inhibited GVBD in vitro and promoted the accumulation of cAMP in a dose-dependent manner irrespective of whether the oocytes were treated for a short duration (2 hr) or for a long duration (36 hr). Evaluation of time course response to 1 mM IBMX or 1 mM theophylline revealed that cAMP levels increased at all the time points when compared with their respective controls and blocked maturation. In contrast, 1 microgram/ml 17 alpha,20 beta-DP not only induced oocyte maturation but also caused an immediate decrease in cAMP within the first 2 hr (from 3.2 +/- 1.3 to 1.3 +/- 0.1 pmol/oocyte) of incubation which was maintained till the end of experiment (36 hr). Likewise, a significant inhibition of GVBD and accumulation of cAMP was recorded even in oocytes pre-stimulated with 1 microgram/ml 17 alpha,20 beta-DP for 6 hr and then treated with different concentrations of IBMX or theophylline. Taken together, these data strongly suggest that in C. batrachus a decrease of oocyte cAMP concentration is a prerequisite for the induction of oocyte maturation, and its increase is associated with the maintenance of meiotic arrest.     

Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation in vivo and in vitro

Heterologous intercellular communication was determined qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites and electrical current in hamster oocyte-cumulus complexes during meiotic maturation in vitro and in vivo. In addition, changes in cell resting potentials during maturation were recorded. Significantly less time was required for germinal vesicle breakdown (GVBD) in oocytes matured in vitro than in oocytes stimulated in vivo (1.81 +/- 0.06 hr, N = 13 vs 2.46 +/- 0.07 hr, N = 18, respectively, P less than 0.001). Resting potentials of the oocyte (RP-o) and cumulus cells (RP-c) significantly increased contemporaneously with GVBD in vitro (RP-o: from -18.9 +/- 3.2 mV to -33.2 +/- 2.9 mV, P less than 0.001; RP-c: from -16.3 +/- 1.9 mV to -27.5 +/- 2.6 mV, P less than 0.001) and in vivo after hCG injection (RP-o: from -16.8 +/- 5.9 mV to -30.1 +/- 3.9 mV, P less than 0.001; RP-c: from -15.5 +/- 3.8 mV to -26.3 +/- 3.2 mV, P less than 0.001). RP-o and RP-c progressively increased with time of culture up to 7 hr (maximum time examined) while the values reached maxima in in vivo matured oocytes 4.5 hr post-hCG and subsequently declined concomitant with the onset of cumulus expansion. Cumulus to oocyte coupling decreased progressively with time after release from meiotic arrest both in vitro and in vivo, as assessed by a progressive reduction in transfer of either uridine marker or lucifer yellow from the cumulus cell to the oocyte. By 4.5 hr after hCG injection, cumulus expansion had begun in 100% of complexes examined. Expansion was extensive by 7 hr post-hCG and spread of lucifer yellow from a cumulus cell was limited to very few adjacent cumulus cells. Oocyte to cumulus cell metabolic coupling also decreased progressively with time in both treatment groups. Examination of the extent of heterologous ionic coupling revealed that ionic coupling exhibited biphasic and, bidirectionally parallel, increases during meiotic maturation. While these temporal changes were observed in both groups, the coupling ratios were much greater in those complexes matured in vitro than in vivo. These results show that dye, metabolic, and electrical coupling exist between the immature hamster oocyte and its surrounding cumulus cells but that during the early stages of meiosis, metabolic and dye coupling decrease, while electrical coupling increases biphasically.     

Induction of mouse oocyte maturation in vivo by perturbants of purine metabolism

Previous studies have suggested a critical role for purines in the maintenance of mouse oocytes in meiotic arrest. If so, then disrupting specific purine metabolic pathways in vivo might induce the resumption of oocyte maturation. To test this hypothesis, immature mice were primed with pregnant mare's serum gonadotropin and 24 h later received, by i.p. injection, one of several drugs that inhibit specific enzymes in the purine metabolic pathways. Cumulus cell-enclosed oocytes were isolated from the ovaries at varying times after drug treatment and assessed for germinal vesicle breakdown (GVBD). The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid (MA) and bredinin (Br), each induced GVBD in a dose-dependent fashion, Br being the more effective agent. When the kinetics of oocyte maturation were examined, 71% of the oocytes from MA-treated mice had undergone GVBD 21-22 h after drug administration. Moreover, 100% GVBD was observed in oocytes from Br-treated mice after 6 h. The action of these drugs appeared to be a direct one and not mediated through stimulation of pituitary gonadotropin release or atresia. Azaserine, an inhibitor of de novo purine synthesis, also induced GVBD in a dose-dependent manner. However, sodium hadacidin and dl-alanosine, inhibitors of adenylosuccinate synthetase, failed to elicit a maturational response in oocytes in vivo. These data support an essential role for guanyl and/or xanthyl derivatives in the maintenance of meiotic arrest in vivo.     

Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effect of mammalian pituitary hormones on germinal vesicle breakdown in oocytes of major carps, Labeo rohita, Cirrhinus mrigala, Catla catla and Cyprinus carpio

Among all the mammalian pituitary hormones, luteinizing hormone (LH) was the most potent in vitro inducer of oocyte maturation in L. rohita, C. mrigala, C. catla and C. carpio. It induced significant germinal vesicle breakdown (GVBD) at concentrations of 10, 1, 0.1 and 0.01 micrograms/ml. At the highest concentration used, LH induced 77.9 +/- 5.9, 73.8 +/- 4.6, 50.3 +/- 2.8 and 40.8 +/- 1.4% GVBD in oocytes of L. rohita, C. mrigala, C. catla and C. carpio, respectively. Among other hormones, follicle stimulating hormone (FSH) induced only a marginally significant GVBD (13.2 +/- 0.8%) in the oocytes of C. carpio, but not in other three species. Thyroid stimulating hormone (TSH), growth hormone (GH) and prolactin (PRL) had no effect on GVBD.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of LH-induced and spontaneous meiotic maturation in mouse oocytes by N alpha-tosyl-L-lysine chloromethylketone

The effect of N alpha-tosyl-L-lysine chloromethylketone (TLCK), an inhibitor of trypsin-type proteases, on luteinizing hormone (LH)-induced and spontaneous meiotic maturation and follicular production of cAMP in mice was determined. When follicle-enclosed mouse oocytes were incubated with LH (1 micron/ml), they underwent the breakdown of the germinal vesicle (GVBD). TLCK (0.02-0.5 mM) inhibited LH-induced GVBD in folliculated oocytes. The concentration (0.5 mM) of TLCK that inhibited LH-induced GVBD did not significantly suppress LH-induced cAMP production by follicle cells. The effect of TLCK on spontaneous maturation in cumulus cell-enclosed and denuded oocytes was also determined. TLCK strongly inhibited spontaneous maturation in denuded oocytes only if it was added to the incubation medium for 1-3 h before oocytes were liberated from the follicular tissue. The inhibition of oocyte maturation by TLCK was significantly greater in cumulus cell-enclosed oocytes than in denuded oocytes, either with or without preincubation with TLCK. These results suggest that trypsin-type protease in oocytes participates in the process of meiotic maturation in mouse oocytes.     

Inhibitory effect of purines in meiotic maturation of denuded mouse oocytes.

The potential action of purines, such as hypoxanthine and adenosine, in meiotic arrest was examined using denuded mouse oocytes. The spontaneous meiotic maturation of denuded oocytes was significantly inhibited by hypoxanthine and/or adenosine in a dose-dependent manner. Germinal vesicle breakdown (GVBD) was inhibited even at a low concentration (1 nM) of hypoxanthine, when hypoxanthine was microinjected into the cytoplasm of denuded oocytes. This inhibitory action was potentiated by co-injection with allopurinol, a metabolic blocker of hypoxanthine that can block a metabolic pathway to uric acid. By contrast, a microinjection of adenosine was no longer effective in inhibiting GVBD. Inhibitory action of purines in meiotic maturation was correlated with sustaining intracellular cAMP levels. GVBD was resumed by econazole, one of the nitroimidazole derivatives which act as inhibitors of catalytic subunit of adenylate cyclase. This compound was effective in counteracting the effect of adenosine, but not the action of 3-isobutyl-1-methylxanthine (IBMX) on GVBD, indicating that adenosine is probably exerted at the level of oocyte plasmalemma. These data suggest that the inhibitory action of hypoxanthine and adenosine in oocyte meiotic maturation may be involved in the regulation of cAMP metabolism in a differential manner.     

Forskolin and mouse oocyte maturation in vitro

Oocytes isolated from mature follicles undergo spontaneous maturation when cultured in vitro. Forskolin, an adenylate cyclase stimulator, inhibited resumption of meiosis of cumulus-free mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 85% of the oocytes treated by forskolin at concentrations of 20 micrograms/ml and higher. The inhibiting effect of forskolin was dose-dependent and reversible. FSH, LH, FSH plus LH, estrogen, progesterone, and estrogen plus progesterone did not reverse the block induced by forskolin in cumulus-free and cumulus-enclosed oocytes. The present results suggest that intracellular cAMP may play a role in the regulation of oocyte maturation.     

Transforming growth factor-alpha augments meiotic maturation of cumulus cell-enclosed mouse oocytes

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor-alpha (TGF-alpha) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell-enclosed oocytes were exposed to TGF-alpha with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denuded oocytes were examined for GVBD following exposure to HX and TGF-alpha. When cumulus cell-enclosed oocytes were exposed to TGF-alpha (1 microgram/ml) in the presence of HX (4 mM), an increase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF-alpha and HX as compared to 33% with HX only. Concentrations of TGF-alpha as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF-alpha, but without HX, was also enhanced. The stimulation of GVBD by TGF-alpha showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF-alpha in the presence of HX, no effect was observed. Our results suggest that TGF-alpha is a potent stimulator of mouse oocyte maturation in vitro and that its effect is mediated by the surrounding cumulus cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Induction and inhibition of amphibian (Rana pipiens) oocyte maturation by protease inhibitor (TPCK)

We report for the first time that oocyte nuclear and cytoplasmic maturation are triggered in vitro in non-hormone-treated amphibian (Rana pipiens) ovarian follicles following transient exposure to synthetic chymotrypsin inhibitor Nα-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The mechanism of action of TPCK in regulating oocyte maturation was investigated and compared to that induced by progesterone or pituitary hormone. Follicular oocytes failed to mature following continuous exposure to the same doses of TPCK in the presence or absence of progesterone. Continuous treatment of follicles with lower levels of TPCK occasionally induced GVBD in the absence of progesterone and augmented maturational effects of low levels of progesterone. TPCK induced maturation of intrafollicular oocytes without stimulating progesterone production and also induced maturation of naked oocytes. Stimulation of follicular progesterone synthesis following gonadotropin stimulation or addition of pregnenolone was inhibited by TPCK, indicating that TPCK affects metabolic processes in both the somatic and germinal components of the ovarian follicle. Oocyte maturation induced by either TPCK or progesterone was inhibited by cycloheximide, calcium-deficient medium, and forskolin. Results suggest that TPCK induces oocyte maturation independent of steroidogenesis via mechanisms similar to those triggered by progesterone involving protein synthesis, formation of cytoplasmic maturation-promoting factor (MPF), and changes in cAMP levels. Our data indicate that a chymotrypsin-like protease plays a role(s) in regulating the oocyte meiotic maturation process.     

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.