Meiosis activating sterol (MAS) regulate FSH-induced meiotic resumption of cumulus cell-enclosed porcine oocytes via PKC pathway

Meiosis activating sterol (MAS) have been found to be able to promote oocytes meiotic maturation of small animals in vitro, such as mouse, rat and rabbit. But in large animals, whether MAS play the same function, especially the physiological mechanisms of MAS on oocytes maturation are not clear. To our knowledge, this is the first time to investigate the role and signal pathway of MAS on FSH-induced porcine oocytes meiotic resumption. Porcine cumulus-enclosed oocytes (CEOs) isolated from 3 to 5mm follicles were cultured in the FSH-medium for 24h supplemented with 0-50 microM RS21745 or 0-100 microM RS21607 (two specific inhibitors of lanosterol 14alpha-demethylase that converts lanosterol to FF-MAS), or cultured in FSH-medium with 25 microM RS21745 for 0-24h firstly, then transferred into a new FSH-medium (the total culture time is 24h). The results revealed that RS21745 or RS21607 could inhibit FSH-induced porcine CEOs meiotic resumption in a dose and time-dependent manner. Meanwhile, FSH-induced cumulus expansion could also be inhibited dose-dependently by RS21745 or RS21607. Otherwise, AY9944-A-7, an inhibitor of Delta14-reductase which promotes cholesterol accumulation from FF-MAS, had no effect on both denuded oocytes (DOs) cultured for 24 or 44 h and CEOs cultured for 24h meiotic resumption, but it could promote CEOs meiotic resumption after 44 h culture. In addition, we got that 10(-8) to 10(-6)M PMA, an activator of PKC pathway, could reverse the inhibiting effect of RS21745 on FSH-induced CEOs meiotic resumption and enhance the rate of germinal vesicle breakdown (GVBD) of CEOs cultured in medium with hypoxanthine (HX). Moreover, 5-10 microM chelerythrine chloride, an inhibitor of PKC, could enhance the inhibitory effect of RS21745 on FSH-induced porcine oocytes resumption of meiosis. All the data of this study support that endogenous FF-MAS takes part in the FSH-induced porcine oocytes meiotic resumption and might play an active role via PKC signal pathway.     

Effects of amphotericin B and ketoconazole on mouse oocyte maturation: implications on the role of meiosis-activating sterol

Meiosis-activating sterol (MAS) has been shown to induce mouse oocytes cultured in the presence of hypoxanthine (HX) to resume meiosis. The present research was conducted to determine whether amphotericin B or ketoconazole (a promoter and an inhibitor of production of MAS), affected oocyte maturation. Mouse cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) were cultured for 24 h in the presence of 4 mM HX with FSH or amphotericin B or ketoconazole. At the end of the culture, the frequency of germinal vesicle break down (GVBD) and polar body formation (PB) were recorded. The results demonstrated: (i) FSH (10-200 IU/l) induced dose-dependent oocytes maturation in CEO, but was without effect on DO. A maximum increase in GVBD and PB was observed with 25-50 IU/l FSH. The presence of FSH (50 IU/l) for 1 h was sufficient to induce meiotic resumption, which after 2 h reached a plateau similar to that of a continuous presence of FSH. (ii) CEO exposed to amphotericin B (0.0025-2.5 microg/l) underwent GVBD dose-dependently, whereas no effect was observed on DO. The presence of amphotericin B (0.025 microg/l) for 1 h stimulated oocyte resumption in a way similar to that of FSH. (iii) Amphotericin B (0.025 microg/l) and FSH (50 IU/l) did not show any additive effect on resumption of meiosis. (iv) Ketoconazole (10(-7)-10(-3) M) inhibited the effect of FSH on resumption of meiosis, but had no effect on oocyte spontaneous maturation. These results show that FSH and amphotericin B induce resumption of meiosis and indicate that they are likely to cause an accumulation of meiosis activating sterols in the CEO, but ketoconazole blocks the production of MAS. The present study supports the notion that MAS plays a physiological relevant role in triggering resumption of meiosis in mouse oocytes.     

Nitric oxide influences the maturation of cumulus cell-enclosed mouse oocytes cultured in spontaneous maturation medium and hypoxanthine-supplemented medium through different signaling pathways

Nitric oxide (NO) has been recently shown to act with a dual action in mouse oocyte meiotic maturation depending on its concentration, but the mechanism(s) through which it influences oocyte maturation has not been fully clarified to date. The purpose of this study was to test the hypothesis that different signaling mechanisms exist for NO-stimulated and NO-inhibited in vitro maturation of meiosis in cumulus cell-enclosed oocytes (CEOs) from PMSG-primed immature female mice. CEOs were cultured in both spontaneous maturation model and hypoxanthine (HX) arrested model to investigate the mechanism(s). Sodium nitroprusside (SNP, an NO donor) at a concentration of 1mM delayed significantly germinal vesical breakdown (GVBD) during the first 5 h of incubation period and further inhibited the formation of first polar body (PB1) at the end of 24 h of incubation. While SNP, at a concentration of 10 microM, stimulated significantly the meiotic maturation of oocytes by overcoming the inhibition of HX. Methinine blue (MB, 10 microM) or 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 microM)), two soluble guanylate cyclase (sGC) inhibitors, could reverse SNP-inhibited spontaneous oocyte maturation, but had no effect on SNP-stimulated meiotic maturation in the presence of HX. 8-Br-cGMP (1mM), a cell-permeating cGMP analogue, demonstrated a significant inhibitory effect on both spontaneous meiotic maturation and HX-arrested meiotic maturation. The delay effect of SNP on GVBD occurrence was similar to that of forskolin (6 microM, an adenylate cyclase stimulator) and rolipram (250 microM, a phosphodiesterase 4 inhibitor), two cAMP elevating reagents. Both forskolin and rolipram reversed significantly the SNP-stimulated meiotic maturation, but did not reverse the SNP-inhibited spontaneous meiotic maturation. Cilostamide (1 microM), the selective inhibitor of phosphodiestrase 3 (PDE3), could mimic the inhibitory effect of HX on the spontaneous meiotic maturation in CEOs and this inhibitory effect could also be reversed by SNP (10 microM). Moreover, sphingosine (3 microM), a protein kinase C (PKC) inhibitor, blocked the SNP-inhibited spontaneous meiotic maturation, but did not block the SNP-stimulated meiotic maturation. Clearly, these results suggest that pathway differences are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation of mouse oocytes.     

Epidermal growth factor receptor activation by protein kinase C is necessary for FSH-induced meiotic resumption in porcine cumulus-oocyte complexes

It is proved that epidermal growth factor (EGF)-like factors mediate gonadotropin-induced rodent oocyte maturation via EGF receptor (EGFR). However, the detail kinetics and signal pathway between FSH and EGF/EGFR is not clear in large animals. In the present study, we investigated the roles of EGFR and protein kinase C (PKC) in FSH-induced porcine oocyte meiotic resumption. Porcine cumulus-oocyte complexes were cultured in NCSU37 medium containing 10% porcine follicular fluid and germinal vesicle breakdown (meiotic resumption) was detected after different treatments. The results showed that EGF-like factor amphiregulin (AR) and EGFR mRNA were expressed in porcine cumulus cells, but not oocytes. FSH significantly induced AR mRNA expression with maximum at 4 h and activated EGFR phosphorylation at 8 h. AR (1-100 ng/ml) dose-dependently induced meiosis resumption of porcine oocyte. The specific EGFR inhibitor, AG1478, but not AG43 (the inactive analog of AG1478), completely blocked FSH, EGF, and AR-induced oocyte meiotic resumption; the inhibitory effect of AG1478 on FSH action gradually decreased when the inhibitor was added at 6 h or later and disappeared when it was added at 11 h; EGF reversed the inhibitory effect on FSH when AG1478 was added within 6 h. FSH triggered porcine oocyte meiotic resumption (at 20 h) later than that of EGF and AR (at 18 h). All these results supported that endogenously produced EGFR activator(s), possibly AR (maximum at 4 h) and EGFR activation (began at 6 h and finished within 11 h), in cumulus cells is necessary for FSH-induced porcine oocyte meiotic resumption (began at 18 h). Furthermore, PKC activator PMA mimicked but PKC inhibitor chelerythrine chloride inhibited FSH action, and AG1478 also suppressed PMA-induced porcine oocyte meiotic resumption. These data together suggested that EGFR activation, by PKC signal pathway, participates in FSH-induced porcine oocyte meiotic resumption.     

Are steroids obligatory mediators of luteinizing hormone/human chorionic gonadotropin-triggered resumption of meiosis in mammals?

Steroids mediate the gonadotropic stimulus of oocyte maturation in fish and amphibians. Such a role of steroids in mammals has not been confirmed until recently. A series of studies presented data suggesting that steroids might be involved in meiosis of mouse oocytes. Here we examined this suggestion using in vitro cultures of rat and mouse follicle-enclosed oocytes (FEOs) and cumulus-enclosed oocytes (CEOs). In FEOs that mature only in response to gonadotropins or other stimuli, we tested the ability of steroids to trigger meiosis and whether addition of steroid receptor antagonists blocks LH/human chorionic gonadotropin stimulation of meiosis. In CEOs that mature spontaneously, we tested whether steroid antagonists block maturation and whether steroids overcome the inhibition of maturation by hypoxanthine (Hx), a mild inhibitor of meiotic resumption. The progesterone antagonists mifepristone (RU 486) and Organon 31710 as well as the estrogen antagonist faslodex did not prevent LH-triggered maturation of rat or mouse FEOs or the spontaneous maturation of CEOs. In accordance, the progesterone agonist promegestone (R5020) and estradiol did not stimulate the resumption of meiosis in rat and mouse FEOs, and both did not overcome the Hx inhibition of meiosis in rat and mouse CEOs. Flutamide, an androgen antagonist, did block meiosis in rat FEOs, but this action could not be affected by adding dihydrotestosterone, suggesting that it was not androgen receptor mediated. Flutamide did not affect spontaneous maturation of rat CEOs, and dihydrotestosterone could not stimulate meiosis inhibited by Hx. Thus, in contrast to lower vertebrates, in mammals, steroids do not seem to serve as an obligatory signal by which the somatic cells of the follicle transfer the gonadotropic stimulation of meiosis to the oocyte.     

Stimulatory role of cyclic adenosine monophosphate as a mediator of meiotic resumption in rabbit oocytes.

The present study was undertaken to assess the role of alterations of intraoocyte cAMP concentrations in the meiotic maturation of isolated and follicle-enclosed oocytes. In isolated oocyte culture, the intracellular cAMP content of denuded oocytes declined within 15 min of incubation, whereas the cAMP content of cumulus-enclosed oocytes did not change substantially for 1.5 h of incubation, and then declined abruptly. Commitment to meiotic maturation was preceded by reduced concentrations of intraoocyte cAMP. Forskolin inhibited the spontaneous maturation of cumulus-enclosed oocytes in a dose-dependent manner. However, this inhibition was attenuated as the duration of incubation increased. Forskolin significantly stimulated the intracellular cAMP content of denuded and cumulus-enclosed oocytes, but intraoocyte cAMP returned to pretreatment values within 4 h. The decline in intraoocyte cAMP was followed by the meiotic maturation of isolated oocytes. In in vitro perfused rabbit ovaries, exposure to forskolin at 10(-4) M, as well as to 50 IU human CG, accelerated the meiotic maturation of follicle-enclosed oocytes. The intraoocyte cAMP content increased significantly within 30 min and reached its maximum 2 h following exposure to forskolin. Thereafter, cAMP decreased abruptly and returned to pretreatment levels by 6 h. These alterations of intraoocyte cAMP contents following exposure to forskolin paralleled those observed in human CG-treated ovaries. The decline in cAMP content of follicle-enclosed oocytes was followed by their meiotic maturation. In conclusion, the sustained elevation of intraoocyte cAMP levels inhibits the initiation of meiotic maturation in isolated and follicle-enclosed oocytes. Within the follicle, resumption of meiosis is triggered via a transient increase in intraoocyte cAMP, but commitment to meiosis must await the decline of intraoocyte cAMP.     

Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18alpha-glycyrrhetinic acid (alpha-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20beta-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.     

Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways

Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 5'-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4-23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocyte maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4-23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4-23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein(s).     

Roles of gonadotropins and meiosis-activating sterols in meiotic resumption of cultured follicle-enclosed mouse oocytes

This study describes a model for short-term culture of intact mouse follicles under serum-free conditions. Follicles were either obtained from immature mice receiving no ovarian stimulation (i.e. no eCG-primed protocol, group I) or from mice undergoing ovarian stimulation (i.e. eCG-primed protocol, group II). Follicles were grouped according to size (100-170, 180-200, 210-250, 260-350 and 360-400 microm, respectively) and cultured for 24h (group I) or for only 6h (group II). Induced meiotic resumption of follicle-enclosed oocytes were evaluated following stimulation with gonadotropins (i.e. FSH and hCG), AY9944-A-7, an inhibitor of Delta14-reductase, and RS-21745, an inhibitor of lanosterol 14alpha-demethylase; both enzymes affect synthesis of the meiosis activating sterols (MAS) that induce oocyte maturation. The frequency of oocyte degeneration was also recorded. In group I, FSH (10-200 IUl-1) and AY9944-A-7 (5, 25 and 50 microM) separately induced resumption of meiosis in oocytes derived from follicles with a diameter of 180-400 microm. hCG (1.0 and 10 IUml-1) exhibited a similar but weaker effect on oocytes present in follicles with a diameter of 260-400 microm. Irrespective of follicular diameter oocytes obtained from follicles in group II responded to hCG and FSH by resuming meiosis. FSH (50 IUl-1) alone or hCG (10 IUml-1) alone both increased the GVBD percentage of oocytes enclosed in follicles with a diameter 260-400 microm, but the response to hCG was not significant compared to control. FSH (50 IUl-1) combination with hCG (10 IUml-1) showed an additive effect raising the rate of GVBD after 6h culture. Addition of 50 or 100 microM RS-21745 was able to attenuate gonadotropins-induced resumption of meiosis to below background levels. In conclusion, the ability of FSH to induce meiotic resumption of follicle-enclosed mouse oocytes is correlated to follicle size, being most pronounced in larger follicles. hCG caused a similar but less pronounced effect. The ability of RS-21745 to inhibit and the ability of AY9944-A-7 to enhance oocyte maturation of follicle-enclosed oocytes support the concept of FSH employing MAS as a downstream signal transduction molecule for initiation of oocyte maturation in mice.     

Oocyte maturation inhibitor

The preovulatory surge of gonadotropins induces within the mature Graafian follicle a series of changes culminating in the release of a fertilizable ovum. These include resumption of the meiotic division, a process held in abeyance from a short time after birth, and the progression of the oocyte from the dictyate stage to the metaphase of the second meiotic division. Here the role of a follicular factor, oocyte maturation inhibitor (OMI), in preventing resumption of meiosis by ova of antral follicles prior to the surge of gonadotropins has been reviewed. The suggested involvement of OMI in regulation of meiosis is based on the following observations: (1) fully grown mammalian oocytes explanted from their follicles undergo meiotic maturation spontaneously, whereas follicle-enclosed ova remain immature until stimulated; (2) co-culture of oocytes isolated from their follicles with follicular granulosa cells, granulosa cell extract and follicular fluid inhibits the spontaneous maturation; (3) the inhibition of oocyte maturation by OMI is reversible and in several of the models employed can be removed by the addition of the physiological trigger of meiosis, luteinizing hormone (LH). The current state of OMI characterization and purification has been described and the involvement of additional factors, such as cyclic AMP, in the regulation of meiosis discussed.     

An inhibitory influence of porcine vasoactive intestinal peptide upon mouse oocyte meiosis in culture

The germinal vesicle (GV) of follicle-enclosed oocytes in mice remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV, suggesting that factors in the follicular constituents sustain the meiotic arrest of oocytes. Vasoactive intestinal peptide (VIP) was demonstrated in the ovary and found to have a wide range of biological effects. In this study, the possibility of VIP to be a factor which induces meiosis-arrest of oocytes was evaluated. Porcine VIP inhibited resumption of meiosis of cumulus-enclosed mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 50% of the oocytes treated by VIP at a concentration of 30 microM. The inhibiting effect of VIP was dose-dependent and reversible. Spontaneous resumption of meiosis of isolated oocytes in vitro was reported to be inhibited by dibutyryl cAMP (dbcAMP) added to the medium, indicating that meiotic arrest can be sustained by maintaining intra-oocyte cAMP above a critical level. It was further reported that follicular fluid contains substances that maintain meiotic arrest in association with exogenous dbcAMP. In the present study, the blocking activity of dbcAMP for the spontaneous resumption of meiosis was not potentiated by the addition of VIP in the medium. The present results suggest that VIP may play a role in the regulation of resumption of oocyte meiosis, and that VIP is not a substance which maintains meiotic arrest in association with cAMP.     

Are steroids dispensable for meiotic resumption in mammals?

Meiosis of vertebrate oocytes is a protracted process initiated within differentiated oocytes before the first meiotic arrest of the first meiotic division. Meiosis normally resumes in response to the stimulation of ovulation, proceeding to metaphase of the second meiotic division. In fish and amphibian oocytes, this resumption is triggered by follicular steroids. By contrast, the role of steroids in the resumption of mammalian oocyte maturation is less clear. Specifically, mammalian meiotic maturation proceeds undisturbed even when steroid production is severely suppressed. This puzzling mammalian divergence has been reexamined recently. Here, we review the published data and conclude that steroids are not necessary for the resumption of mammalian meiosis. Nevertheless, steroids are probably involved in follicular growth, somatic-cell differentiation and the acquisition of developmental competence of mature ova.     

Role of meiosis activating sterols, MAS, in induced oocyte maturation

Meiosis of follicle enclosed oocytes is maintained in the prophase of the first meiotic division and oocytes do not spontaneously resume meiosis during oocyte growth and follicle development. Arrest of the meiotic process is most likely secured by the presence of follicular purines, e.g. hypoxanthine, which maintain high levels of cAMP in the oocyte and which also in vitro prevent oocytes from resuming meiosis. Only in response to the mid-cycle surge of gonadotropins will oocytes of preovulatory follicles overcome the meiosis arresting effect of hypoxanthine and resume meiosis proceeding to the metaphase of the second meiotic division. Morphologically, resumption of meiosis is observed by the disappearance of the oocyte's nuclear membrane (germinal vesicle), a process called germinal vesicle breakdown (GVB). The molecular mechanism down-stream to receptor activation by which the mid-cycle surge of gonadotropins induces oocytes to resume meiosis is, however, only partly understood. The oocyte itself lacks gonadotropin receptors and its action is mediated through the attached cumulus cells. In vitro it has been shown that FSH induces synthesis of a signal in the cumulus cells, which overcomes the meiosis arresting effect of hypoxanthine. We have shown that a group of sterols, meiosis activating sterols (MAS), induces oocyte maturation in vitro even in oocytes depleted of cumulus cells. MAS were identified as intermediates in the cholesterol biosynthesis between lanosterol and cholesterol. The two best characterized members of the MAS family are FF-MAS purified from human follicular fluid (4,4-dimethyl-5alpha-cholest-8,14,24-triene-3beta-ol) and T-MAS purified from bull testicular tissue (4,4-dimethyl-5alpha-cholest-8,24-diene-3beta-ol). The synthesis, quantification, localization and tissue-accumulation of MAS are reviewed. Several publications have documented the pharmacological effect of MAS in different species, including oocytes from mouse, rat and human. Conflicting results obtained by the use of sterol synthesis inhibitors, which prevent MAS-accumulation, are also discussed. Whether FSH actually uses MAS as a signal transduction molecule for inducing oocyte maturation and the mechanism by which MAS induce resumption of meiosis is currently unknown, but data to support that MAS is part of the FSH induced signal transduction pathway are presented.     

Dissociation between the inhibitory and the stimulatory action of cAMP on maturation of rat oocytes

The possible mediatory role of cAMP in the induction of oocyte maturation by luteinizing hormone (LH) is not yet clear since evidence for both inhibitory and stimulatory actions of the nucleotide on the oocyte has been provided. To elucidate the role of cAMP in regulation of oocyte meiosis we tried in the present study to dissociate between the inhibitory and stimulatory action of this nucleotide on oocyte maturation. To induce maturation, oocytes enclosed by their follicles were transiently exposed to either dibutyryl cAMP (dbcAMP) or to the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Inhibition of maturation was obtained by the addition of the above agents to either follicle-enclosed oocytes incubated in the presence of LH or isolated cumulus-free oocytes that mature spontaneously in vitro. We found that inhibition of oocyte maturation is obtained by a relatively low dose of either dbcAMP or MIX while higher concentrations of these agents are required to induce oocyte maturation. Coupling of the oocyte to the cumulus cells, as expressed by the fraction of labeled uridine transferred from the cumulus cells to the oocyte following exposure of the follicle-enclosed cumulus-oocyte complex to MIX, was also determined. We found that uncoupling of the oocyte from the cumulus cells corresponded with the induction, but not inhibition of oocyte maturation, both by its concentration dependence and time-course. We suggest that cAMP has a dual role in regulation of oocyte maturation. Lower levels of the nucleotide act to maintain meiotic arrest, while elevated levels of cAMP mediate LH action to induce meiosis resumption.     

Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A

Progesterone induces G2-arrested Xenopus oocytes to develop into fertilizable eggs in a process called meiotic maturation. Protein kinase A (PKA), the cAMP-dependent protein kinase, has long been known to be a potent inhibitor of meiotic maturation, but little information is available on how PKA functions. We have cloned two Xenopus PKA catalytic subunit isoforms, XPKAalpha and XPKAbeta. These proteins are 89% identical and both inhibit progesterone-induced meiotic maturation when overexpressed at low levels, suggesting that PKA activity is tightly regulated in the oocyte. Unexpectedly, catalytically inactive XPKA mutants are able to block progesterone-induced maturation as efficiently as the wild-type active XPKA. These mutants also block meiotic maturation induced by Mos, but are less efficient at inhibiting Cdc25C-induced maturation. Our results indicate that PKA can inhibit meiotic maturation by a novel mechanism, which does not require its kinase activity and is also independent of binding to the PKA regulatory subunits.     

Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker.

A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown/maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker.     

Cyclic adenosine 3',5'-monophosphate-dependent activation of mitogen-activated protein kinase in cumulus cells is essential for germinal vesicle breakdown of porcine cumulus-enclosed oocytes

MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.     

Cellular, biochemical and molecular mechanisms regulating oocyte maturation

The original model for regulation of oocyte maturation proposed by us in 1978 postulated that gap junction-mediated transmission of cAMP from the follicle cells to the oocyte inhibits meiosis and that luteinizing hormone (LH) terminates the flux of the follicle cAMP to the oocyte. A decrease in oocyte cAMP below inhibitory threshold occurs since oocytes lack the ability to generate sufficient amounts of cAMP to compensate for the phosphodiesterase activity. Our previous studies provided evidence to support this model. More recent studies in our laboratory were directed at identification of the cellular biochemical and molecular events initiated within rat oocytes upon the relief of cAMP inhibition. These studies: (i) identified an oocyte specific A kinase anchoring protein (AKAP) that is phosphorylated in oocytes resuming meiosis, (ii) confirmed that cdc25B governs meiosis reinitiation and demonstrated that its expression is translationally regulated, (iii) substantiated the indispensable role of proteasomal degradation at completion of the first meiotic division in a mammalian system, (iv) elucidated the role of MPF reactivation in suppressing interphase between the two meiotic divisions and (v) provided evidence that mos translation is negatively regulated by a protein kinase A (PKA)-mediated action of cAMP and is dependent on an active MPF. A detailed account on each of these findings is presented in this chapter.     

Role of cyclic nucleotide signaling in oocyte maturation

The development of the ovarian follicle, oocyte maturation, and ovulation require a complex set of endocrine, paracrine, and autocrine inputs that are translated into the regulation of cyclic nucleotide levels. Changes in intracellular cAMP mediate the gonadotropin regulation of granulosa and theca cell functions. Likewise, a decrease in cAMP concentration in the oocyte has been associated with the resumption of meiosis. Using pharmacological and molecular approaches, we determined that the expression of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, is compartmentalized in the ovarian follicle of all species studied, with PDE3 present in the oocytes and PDE4s in granulosa cells. The PDE3 expressed in the mouse oocyte was cloned, and the protein expressed in a heterologous system had properties similar to those of a PDE3A derived from somatic cells. Inhibition of the oocyte PDE3 completely blocked oocyte maturation in vitro and in vivo, demonstrating that the activity of this enzyme is essential for oocyte maturation. Heterologous expression of PDE3A in Xenopus oocyte causes morphological changes distinctive of resumption of meiosis (GVBD), as well as activation of mos translation and MAPK phosphorylation. Using mRNA and antibody microinjection in the Xenopus eggs, we have shown that PDE3 is downstream from the kinase PKB/Akt in the pathway that mediates IGF-1 but not progesterone-induced meiotic resumption. The presence of a similar regulatory module in mammalian oocytes is inferred by pharmacological studies with PDE3 inhibitors and measurement of PDE activity. Thus, PDE3 plays an essential role in the signaling pathway that controls resumption of meiosis in amphibians and mammals. Understanding the regulation of this enzyme may shed some light on the signals that trigger oocyte maturation.     

Atrial natriuretic peptide inhibits spontaneous rat oocyte maturation

We report results of experiments demonstrating a dose-dependent inhibition of spontaneous maturation (resumption of meiosis) in rat oocyte-cumulus complexes by atrial natriuretic peptide (ANP). The inhibition was persistent over the time period studied. The ANP analog Tyr8-ANP, which mediates smooth muscle relaxation in other organs without elevating cGMP levels, did not inhibit the spontaneous maturation. ANP, but not Tyr8-ANP, dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes. Furthermore, sodium nitroprusside (SNP), that stimulates a soluble form of guanylate cyclase, inhibited spontaneous maturation in oocyte-cumulus complexes and stimulated cGMP accumulation in oocyte-cumulus complexes. Neither ANP nor SNP stimulated cAMP accumulation. In oocytes where the surrounding cumulus cells had been removed neither ANP nor SNP inhibited the spontaneous maturation. These results demonstrate that cumulus cells, but not the oocyte itself, have ANP receptors and guanylate cyclases. Furthermore, ANP, via cGMP, can influence oocyte meiosis, suggesting a possible involvement of ANP and cGMP in the control of the meiotic process in rat oocytes.