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Role of cyclic nucleotide signaling in oocyte maturation
Molecular and Cellular Endocrinology 187 (2002) 153– 159 www.elsevier.com/locate/mce
Role of cyclic nucleotide signaling in oocyte maturation Marco Conti a,*, Carsten Bo Andersen a, Francois Richard b, Celine Mehats a, Sang-Young Chun c, Kathleen Horner a, Catherine Jin a, Alex Tsafriri d a
Di6ision of Reproducti6e Biology, Department of Gynecology and Obstetrics, Stanford Uni6ersity School of Medicine, Stanford, CA 94305, USA b Centre de Recherche en Biologie de la Reproduction, De´partement des sciences animals, Uni6ersite´ La6al, Ste-Foy, Que., Canada c Hormone Research Center, Chonnam National Uni6ersity, Kwangju, South Korea d The Weizmann Institute of Science, Department of Biological Regulation, Reho6ot 76100, Israel
Abstract 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. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Oocyte maturation; Meiosis; cAMP; Mammalian; Xenopus
1. Meiosis and meiotic competence of the mammalian oocyte Unlike male germ cells that enter meiosis only at puberty, the ontogeny of the mammalian oocyte is characterized by two consecutive phases of meiotic progression/arrest that span the embryonic, fetal, pubertal, and adult life. In most mammalian species, the first meiotic division is initiated during prenatal life or shortly after birth (Eppig, 1993; Tsafriri and Dekel, 1994). Once the transition between prophase and
* Corresponding author. Tel.: + 1-650-725-2452; fax: +1-650-7257102. E-mail address: [email protected] (M. Conti).
metaphase (diplotene stage) is reached (around birth), unknown mechanisms produce a meiotic arrest in the late prophase that is maintained until shortly before ovulation. Rather than continuing in their condensation, chromosomes disperse in the nucleus, and transcription is resumed. In the growing preantral follicle, oocytes remain in this diplotene stage and are incompetent to resume meiosis. This inability to re-enter the cell cycle is probably due to absence, or accumulation below a threshold, of key components necessary for the activation of the maturation promoting factor (MPF) (Kanatsu-Shinohara et al., 2000; Mitra and Schultz, 1996; Moore et al., 1996). With growth of the oocyte in the preovulatory follicle, the components involved in the control of the cell cycle accumulate to a level permissive for meiosis (meiotic competence). Meiotic
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resumption of the oocyte, however, is prevented by inhibitory signals derived from the somatic compartment. In rat, the oocyte will resume meiosis 2– 3 h after the preovulatory gonadotropin surge. The prominent nucleus, characteristic of the dictyate oocyte (germinal vesicle, GV), disappears following chromosome condensation and dissolution of the nuclear membrane GV breakdown (GVBD). Meiosis continues with chromosome segregation, asymmetric division, and extrusion of a polar body, and, unlike mitosis, it proceeds without an interphase and DNA replication to a second division, which is arrested in metaphase II (second meiotic arrest). A cytostatic factor (CSF) (Masui and Market, 1971), of which the mos kinase is an essential component (Sagata et al., 1989), is responsible for maintaining the metaphase II arrest. Only a metaphase II-arrested secondary oocyte is fertilizable by spermatozoa.
2. The follicle environment maintains the oocyte maturational arrest In all mammalian species examined, oocytes that are removed from the follicle undergo spontaneous maturation (GVBD), suggesting that the follicular environment prevents the maturation of the oocyte (Pincus and Enzmann, 1935; Edwards, 1965). In some instances, it has been shown that cumulus oocyte complexes need to physically interact with mural granulosa cells to maintain meiotic arrest (Hillensjo¨ et al., 1979), indicating that intercellular communications between the cells of the follicle, perhaps mediated by gap junctions, are indispensable for maintaining this arrest. In other cases, this physical communication between somatic cells and the oocyte was not necessary because follicular fluid inhibited resumption of meiosis (Tsafriri and Dekel, 1994). These observations have led to the hypothesis that an oocyte meiotic inhibitor (often termed OMI) is produced by granulosa cells. Several candidates have been proposed for this inhibitory activity. Tsafriri et al. have proposed that a peptide of less than 2000 Da is responsible for inhibition of meiosis (Tsafriri and Channing, 1975; Tsafriri et al., 1976; Tsafriri and Dekel, 1994); however, the biochemical nature of this protein remains unknown to date. Several studies indicate that hypoxanthine may fulfill the properties of this inhibitor of oocyte maturation (Eppig et al., 1985; Eppig and Downs, 1987; Downs et al., 1989). Hypoxanthine is present in the follicular fluid at 1– 2 mM, a concentration sufficient to inhibit oocyte maturation (Eppig et al., 1985). Finally, it is possible that cAMP, either produced locally or transferred to the oocyte through gap junctions, may function as the inhibitory signal produced by granulosa cells (Dekel, 1988; see below).
3. The blockade of meiotic resumption is due to elevated cAMP levels in the oocyte Studies in species as diverse as starfish, Xenopus, and mammals have reached the identical conclusion that levels of the second messenger cAMP in the oocyte play a critical role in maintaining meiotic arrest (Ferrell, 1999; Taieb et al., 1997). High cAMP levels in the oocyte maintain protein kinase A (PKA) in an active/ dissociated state which causes phosphorylation of unknown protein substrates (Fig. 1). Classic studies using injections of the PKA inhibitor, PKI, or regulatory and catalytic subunits of PKA in Xenopus oocytes, support this model (Maller and Krebs, 1980, 1977). Similar findings with injections of PKA have been reported for mouse oocytes (Bornslaeger et al., 1986), and pharmacological manipulations to increase cAMP levels in mammalian oocytes invariably produces a blockade of meiosis (reviewed in Conti et al., 1998). Consistent with this hypothesis, it has long been known that phosphodiesterase (PDE) inhibitors, such as the xanthine IBMX, block spontaneous mammalian oocyte maturation (Conti et al., 1998), but the exact PDE target for this pharmacological intervention was unknown until recently (see below). In mammalian oocytes of the preovulatory follicle, how cAMP is maintained above a threshold is still unclear (Fig. 1). Conflicting results have been reported on the expression of the enzyme adenylyl cyclase (AC) responsible for cAMP synthesis, and it is uncertain whether the oocyte is able to produce sufficient cAMP to prevent meiosis (Schultz et al., 1983; Dekel and
Fig. 1. cAMP homeostasis and the meiotic arrest. See details in the test.
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Beers, 1980; Tsafriri and Dekel, 1994). As an alternative hypothesis, it has been proposed that cAMP diffuses from cumulus cells to the oocyte through gap junctions (Dekel, 1988; Eppig, 1991) (Fig. 1). This diffusion, perhaps in synergy with the inhibition of the oocyte PDE by hypoxanthine, maintains cAMP levels high enough to prevent meiotic resumption. Although gap junctions play a critical role in oocyte/cumulus cell communication, as demonstrated by the inactivation of connexin 37 (Simon et al., 1997) by homologous recombination, this latter knockout model does not provide information on the role of gap junctions at the time of meiotic resumption (Carabatsos et al., 2000).
4. PDE3A as an essential component of the signaling pathway that controls the meiotic arrest Our laboratory has identified the major PDE form expressed in rat and mouse oocyte as a PDE3A and found that specific inhibitors for this class of PDEs prevents oocyte maturation in vitro and in vivo (Tsafriri et al., 1996; Wiersma et al., 1998), therefore demonstrating an essential role of this enzyme in meiotic arrest (Fig. 1). The presence of PDE3A in rat and mouse oocytes has been confirmed by biochemical and molecular approaches. Complementary DNAs containing a complete PDE3A open reading frame were retrieved from a mouse oocyte library (Shitsukawa et al., 2001). Expression of the oocyte PDE cDNA was associated with PDE activity with the properties of a PDE3A described in human. Western blot analysis of the recombinant protein demonstrated the expression of a polypeptide of approximately 130 kDa. Polyclonal antibodies have been produced by injecting a GST fused to the carboxyl terminus of the oocyte PDE3A. The recombinant PDE3A is recognized by this antibody (Shitsukawa et al., 2001). The same antibody immunoprecipitates 90% of the PDE activity from mouse or rat oocytes in a specific manner, confirming that a PDE3A is the major PDE expressed in oocytes. Preliminary evidence suggests that a splicing variant of PDE3A, devoid of the membrane anchoring domain, may be expressed in oocytes (Shitsukawa et al., 2001). Taken together, these studies provide biochemical and immunological evidence that PDE3A is expressed and is active in rodent oocytes. Preliminary data obtained with Rhesus macaque oocytes further indicate that PDE3s play an important role in meiotic arrest in primates as well (Jensen et al., 2000). Detailed pharmacological profiling studies to complement the characterization of the PDE3 expressed in rodent oocytes demonstrate that PDE3A controls a pool of cAMP critical for regulation of meiotic resumption (Shitsukawa et al., 2001). Selective and nonselec-
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tive PDE inhibitors were used at a range of concentrations between 10 − 10 and 10 − 3 M in PDE assays using recombinant PDE3A cloned from the oocytes, and PDE from extracts of cumulus enclosed oocytes (COCs). The rank of potency was then compared to the potency of the compounds in inhibiting resumption of meiosis (measured as GVBD). This comparison demonstrated a highly statistically significant correlation between the potency of the compounds in inhibiting recombinant PDE3A, PDE derived from the oocytes, and GVBD. These findings provide further evidence that the PDE that controls the cAMP pool involved in meiosis resumption is a PDE3A. It should be noted that hypoxanthine, which is thought to be a natural inhibitor of meiotic resumption, inhibited the oocyte PDE3A with a potency very similar to that measured for inhibition of meiotic resumption. This in vitro observation confirms and extends in vivo experiments injecting PDE3-specific inhibitors in rat and mice (Wiersma et al., 1998). These inhibitors caused a dissociation between meiotic maturation and ovulation. As a result, ovulated oocytes from PDE3treated rats or mice were still in a GV state and could not be fertilized. Because granulosa cells were not affected by this treatment, this observation provides ‘proof of concept’ for targeting oocyte maturation as an effective method of fertility control (Wiersma et al., 1998). Collectively, the above reviewed data demonstrate that PDE3A plays an essential role in the maintenance in meiotic arrest of the oocyte not only in vitro, but also in vivo. Our findings also show that signals for resumption of meiosis require an active PDE3A.
5. How is meiosis initiated? During the normal ovulatory cycle, the physiological stimulus for meiotic resumption is the LH surge (Tsafriri and Dekel, 1994). Pharmacological blockade of the LH surge with Nembutal, GnRH antagonists, or hypophysectomy prevents oocyte maturation. Similarly, inactivation of the LH receptor is associated with a failure of the oocyte to undergo meiotic resumption and ovulation. Because gonadotropin receptors are not detectable in the oocyte, it has been concluded that LH stimulates granulosa cells and that this stimulation removes the inhibitory constraint on meiotic resumption, perhaps by blocking the production of an inhibitory substance (Fig. 2). Several studies have indicated that oocyte maturation is associated with a decrease in cAMP in both rat (Aberdam et al., 1987) and mouse (Vivarelli et al., 1983; Schultz et al., 1983) oocytes, suggesting that LH signals this decrease in cAMP. These findings are somewhat paradoxical because it is well established that LH pro-
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Fig. 2. Possible mechanisms involved in resumption of meiosis in mammals.
duces a large increase in cAMP in granulosa cells (Richards, 1980; Hsueh et al., 1984). To reconcile these discrepancies, it should be noted that, in spite of the presence of gap junctions, granulosa cells and oocytes act as two separate compartments in terms of cAMP action and an increase in cAMP in the somatic cell compartment is somehow translated into a decrease in cAMP in the oocyte. Such a situation is strongly suggested by our finding that manipulations of cAMP in the two compartments through selective inhibition of PDEs in somatic cells and oocytes produce opposing effects on maturation. Inhibitors of the oocyte PDE3 invariably block oocyte maturation induced by gonadotropins, whereas inhibition of PDE4 in granulosa cells produces meiotic resumption and ovulation in the absence of LH (Tsafriri et al., 1996). Studies with selective PKA inhibitors also are consistent with this hypothesis because activation of the PKA present in granulosa cells stimulates resumption of meiosis, whereas stimulation of the PKA in the oocytes blocks oocyte maturation (Downs and Hunzicker-Dunn, 1995). Gonadotropin-induced severance of the communication between cumulus cells and oocytes may be the trigger for a decrease in cAMP and resumption of meiosis. However, conflicting results have been published on this issue, with several studies indicating that a loss of communication via gap junctions lags behind resumption of meiosis by several hours (Moor et al., 1980; Dekel et al., 1981; Eppig, 1982). The above two possibilities are not mutually exclusive, as closure of gap junctions may cooperate with an active signal, which together decreases cAMP. Some of the data thus far published are also compatible with a more complex model whereby cumulus cells send a positive signal that activates the oocyte, overcoming the inhibitory cAMP levels (Fig. 2). This latter scenario is suggested by findings with an in vitro mouse model that is often used to study oocyte maturation
(Eppig et al., 1983; Freter and Schultz, 1984; Byskov et al., 1997). In this experimental paradigm, cumulus oocyte complexes (COC) are maintained in culture in the presence of hypoxanthine or dbcAMP, and meiotic resumption is induced by FSH, thus demonstrating that cumulus cells can send a positive stimulus to the oocyte that overrides the cAMP inhibitory effects. Stimuli other than the gonadotropin FSH, including EGF, GH, and TNFa, produce a similar effect, probably by acting on cumulus cells (Dekel and Sherizly, 1985; Apa et al., 1994; Feng et al., 1988). However, this stimulation is ineffective with PDE inhibitors more potent and specific than hypoxanthine, or high dbcAMP concentrations. It is, therefore, not clear whether this positive stimulus is effective in the presence of high cAMP levels or whether a decrease in cAMP is obligatory for the resumption of meiosis. Further studies under conditions where only the oocyte cAMP is affected by PDE3-specific inhibitors should provide further insight into the mechanisms underlying this ‘hormone-stimulated’ oocyte maturation. The quest for a positive signal originating from granulosa cells and inducing oocyte maturation has led to the isolation of MAS, a cholesterol derivative found in both testis and ovary (Byskov et al., 1995). FF-MAS is found in the follicular fluid, its concentration is increased by gonadotropins, and in vitro, FF-MAS induces resumption of meiosis of oocytes that are maintained in an arrested state by hypoxanthine (Byskov et al., 1997). Interestingly, differences have been found in the spontaneous and MAS-induced oocyte maturation, and it appears that MAS may be able to overcome the blockade imposed by high cAMP levels. Measurement of PDE present in the oocyte at different times during spontaneous maturation showed a transient increase in activity prior to GVBD (Richard et al., 2001). This increase was blocked by PDE3 inhibitors, but not PDE4, indicating that the oocyte PDE3 is involved in this regulation. Measurement of the PDE activity in oocyte derived from intact follicles incubated in vitro for 2 h with hCG also showed an increase in PDE3 activity. Thus, an increase in oocyte PDE activity precedes both spontaneous and hCG-induced oocyte maturation. These findings support the concept that an active signal is responsible for oocyte maturation and that an increase in PDE activity may be a component of the signaling cascade involved in re-entry into the cell cycle.
6. Which signal transduction pathway of the oocyte triggers GVBD and resumption of meiosis? The above findings are compatible with the hypothesis that oocyte maturation is dependent on the in-
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traoocyte cAMP levels and several signaling pathways are activated in the oocyte to either decrease cAMP levels or to override the cAMP inhibitory effects. The comparison with the Xenopus oocyte may provide important clues on the mechanism of meiotic resumption in mammalian oocytes. Progesterone produced by follicular cells is considered the physiological stimulus for resumption of meiosis in Xenopus oocytes (Maller and Krebs, 1980). Because IGF-1 potently activates resumption of meiosis in the frog, it is possible that the two signals cooperate under physiological conditions. Progesterone acts through a ‘nonnuclear’ steroid receptor to decrease cAMP synthesis by inhibiting adenylyl cyclase, but with unclear effects on PDE activity (Maller and Krebs, 1980; Ferrell, 1999). The regulation of the adenylyl cyclase by progesterone occurs by a mechanism different from the classical Gi-mediated inhibition and may depend on the level of free Gbg (Lutz et al., 2000; Sheng et al., 2001). On the contrary, a PDE activation via a PI3K/PKB regulation plays an essential role in IGF-1 signaling for re-entry into the cell cycle (Andersen et al., 1998; Liu et al., 1995; Muslin et al., 1993). Thus, both stimuli appear to converge in the Xenopus oocytes to produce a decrease in cAMP which, in turn, induces oocyte maturation. Although earlier work suggested the presence of a progesterone receptor exposed on the cell surface, recent data are consistent with the view that the ‘classical’ nuclear progesterone receptor is mediating the steroid effects on meiotic resumption in the Xenopus oocytes (Tian et al., 2000; Bayaa et al., 2000; Maller, 2001). Expression of an active mouse oocytes PDE3A in Xenopus oocytes produced GVBD to the same extent as progesterone or insulin (Andersen et al., manuscript in preparation). It also induced mos translation and ERK phosphorylation/activation, demonstrating that the complete program involved in the re-entry into the cell cycle is inducible by an increase in PDE activity. Moreover, a concentration response study demonstrated that a small increase in PDE activity was sufficient to produce meiotic resumption. Thus, an increase in PDE activity is sufficient to induce the complete pattern of changes that precede GVBD and re-entry into the cell cycle. Several findings, including our own, suggest that the progesterone and IGF-1 signaling pathways may be more integrated than originally thought. Inhibition of PI-3 kinase has some effects on the progesterone stimulation of resumption of meiosis (Muslin et al., 1993), and inhibition of PKB or PDE also interferes with progesterone activation (Andersen et al., manuscript in preparation). Interestingly, it has been recently proposed that steroid receptors are in a complex with PI-3 kinase and occupancy by steroid hormones causes acti-
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vation of this cascade (Simoncini et al., 2000; Maller, 2001), further implicating this pathway in the nongenomic action of steroids and in the control of meiotic maturation. The recent discovery that nuclear progesterone receptors are expressed in Xenopus oocytes is consistent with this view (Bayaa et al., 2000; Tian et al., 2000). Because IGF-1 stimulates resumption of meiosis in oocytes from the Xenopus lae6is via a PI3-K activation, we tested the hypothesis that the serine/threonine kinase PKB/Akt is involved in this process and that a PDE3 activation is distal to this kinase (Fig. 3). The activation of the PKB/Akt is thought to be a critical step in the phosphoinositide 3-OH kinase (PI3-K) pathway that regulates cell growth and differentiation. Injection of mRNA coding for a constitutively active PKB/Akt in Xenopus oocytes induced GVBD to the same extent as progesterone or insulin treatment (Andersen et al., 1998). The induction of GVBD was associated with the phosphorylation of the MAP kinase ERK2, indicating that all the components of the maturation process are activated in the injected oocytes. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a K179M mutation in the catalytic domain that abolishes the kinase activity, had no effect (Andersen et al., 1998). A kinase with a mutation in the myristoylation signal sequence did not induce GVBD, in spite of the high levels of expression. Thus, both the membrane interaction and the kinase activity of PKB/Akt are required to promote resumption of meiosis. The induction of GVBD by insulin/IGF-1 or PKB/Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE3 is required for the Akt action. More importantly, the constitutively
Fig. 3. Putative signaling pathways in regulation of meiosis in Xenopus oocyte.
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active Akt caused a two-fold increase in the PDE activity endogenous to the oocyte (Andersen et al., 1998). Finally, an antibody that blocks the Akt activity also blocks oocyte maturation induced by insulin. These data demonstrate that PKB/Akt is part of the mechanism controlling resumption of meiosis in the Xenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation. Given that PDE3 activity is essential for oocyte maturation in rodents, the comparison with Xenopus suggests that a PI3K/Akt/PDE3A pathway may be an important component in regulating meiotic resumption in mammals as well. In summary, although the exact intracellular signals controlling meiotic resumption in mammalian oocytes remains to be elucidated, homologies with the Xenopus model suggest that multiple pathways may participate in this control. These signaling pathways may converge in the regulation of cAMP levels, perhaps by regulating the PDE3 expressed in the oocytes, thus removing an inhibitory constraint. This decrease in cAMP may be sufficient to trigger meiotic resumption, but it is also possible that signals that override the cAMP inhibitory effect may participate in the control of oocyte maturation. Understanding these mechanisms of control will open new avenues for the pharmacological manipulation of fertility.
Acknowledgements The work from the authors’ laboratory was supported by NIH grant HD20788 and by NICHD/NIH through a cooperative agreement (U54 HD31398) as part of the Specialized Cooperative Centers Program in Reproduction Research.
References Aberdam, E., Hanski, E., Dekel, N., 1987. Maintenance of meiotic arrest in isolated rat oocytes by the invasive adenylate cyclase of Bordetella pertussis. Biol. Reprod. 36, 530 –535. Andersen, C.B., Roth, R.A., Conti, M., 1998. Protein kinase B/Akt induces resumption of meiosis in Xenopus oocytes. J. Biol. Chem. 273, 18705 – 18708. Apa, R., Lanzone, A., Miceli, F., Mastrandrea, M., Caruso, A., Mancuso, S., Canipari, R., 1994. Growth hormone induces in vitro maturation of follicle- and cumulus-enclosed rat oocytes. Mol. Cell. Endocrinol. 106, 207 –212. Bayaa, M., Booth, R.A., Sheng, Y., Liu, X.J., 2000. The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism. Proc. Natl. Acad. Sci. USA 97, 12607 – 12612. Bornslaeger, E.A., Mattei, P.M., Schultz, R.M., 1986. Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev. Biol. 114, 453 – 462. Byskov, A.G., Andersen, C.Y., Nordholm, L., Thogersen, H., Guoliang, X., Wassmann, O., Andersen, J.V., Guddal, E., Roed, T.,
1995. Chemical structure of sterols that activate oocyte meiosis. Nature 374, 559 – 562. Byskov, A.G., Andersen, C.Y., Hossaini, A., Guoliang, X., 1997. Cumulus cells of oocyte – cumulus complexes secrete a meiosis-activating substance when stimulated with FSH. Mol. Reprod. Dev. 46, 296 – 305. Carabatsos, M.J., Sellitto, C., Goodenough, D.A., Albertini, D.F., 2000. Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Dev. Biol. 226, 167 – 179. Conti, M., Andersen, C.B., Richard, F.J., Shitsukawa, K., Tsafriri, A., 1998. Role of cyclic nucleotide phosphodiesterases in resumption of meiosis. Mol. Cell. Endocrinol. 145, 9 – 14. Dekel, N., Regulation of oocyte maturation: the role of cAMP. In: In Vitro Fertilization and other Assisted Reproduction. Acad. Sci., New York, 1988, pp. 211 – 216. Dekel, N., Beers, W.H., 1980. Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus. Dev. Biol. 75, 247 – 254. Dekel, N., Sherizly, I., 1985. Epidermal growth factor induces maturation of rat follicle-enclosed oocytes. Endocrinology 116, 406 – 409. Dekel, N., Lawrence, T.S., Gilula, N.B., Beers, W.H., 1981. Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev. Biol. 86, 356 – 362. Downs, S.M., Daniel, S.A.J., Bornslaeger, E.A., Hoppe, P.C., Eppig, J.J., 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., Hunzicker-Dunn, M., 1995. Differential regulation of oocyte maturation and cumulus expansion in the mouse oocyte – cumulus cell complex by site-selective analogs of cyclic adenosine monophosphate. Dev. Biol. 172, 72 – 85. Edwards, R.G., 1965. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208, 349 –351. Eppig, J.J., 1982. The relationship between cumulus cell –oocyte coupling, oocyte meiotic maturation, and cumulus expansion. Dev. Biol. 89, 268 – 272. Eppig, J.J., 1991. Intercommunication between mammalian oocytes and companion somatic cells. Bioessays 13, 569 – 574. Eppig, J.J., 1993. Regulation of mammalian oocyte maturation. In: Adashi, E.Y., Leung, P.C.K. (Eds.), The Ovary. Raven Press, New York, pp. 185 – 208. Eppig, J.J., Downs, S.M., 1987. The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation. Dev. Biol. 119, 313 – 321. Eppig, J.J., Freter, R.R., Ward-Bailey, P.F., Schultz, R.M., 1983. Inhibition of oocyte maturation in the mouse: participation of cAMP, steroid hormones and a putative maturation-inhibitory factor. Dev. Biol. 100, 39 – 49. Eppig, J.J., Ward-Bailey, P.F., Coleman, D.L., 1985. Hypoxanthine and adenosine in murine ovarian follicular fluid: concentrations and activity in maintaining oocyte meiotic arrest. Biol. Reprod. 33, 1041 – 1049. Feng, P., Catt, K.J., Knecht, M., 1988. Transforming growth factorbeta stimulates meiotic maturation of the rat oocyte. Endocrinology 122, 181 – 186. Ferrell, J.E. Jr., 1999. Xenopus oocyte maturation: new lessons from a good egg. Bioessays 21, 833 – 842. Freter, R.R., Schultz, R.M., 1984. Regulation of murine oocyte meiosis: evidence for a gonadotropin-induced, cAMP-dependant reduction in a maturation inhibitor. J. Cell Biol. 98, 1119 –1128. Hillensjo¨ , T., Channing, C.P., Pomerantz, S.H., Schwartz-Kripner, A., 1979. Intrafollicular control of oocyte maturation in the pig. In Vitro 15, 32 – 39.
M. Conti et al. / Molecular and Cellular Endocrinology 187 (2002) 153–159 Hsueh, A.J., Adashi, E.Y., Jones, P.B., Welsh, T.H. Jr., 1984. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5, 76 – 127. Jensen, J., Schwinof, K., Hazzard, T., Zelinski-Wooten, M., Conti, M., Stouffer, R., 2000. In vitro exposure to phosphodiesterase 3 inhibitors prevents spontaneous resumption of meiosis in Rhesus macaque oocytes. In: Annual Meeting of the Society for the Study of Reproduction, 272pp. Kanatsu-Shinohara, M., Schultz, R.M., Kopf, G.S., 2000. Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes. Biol. Reprod. 63, 1610 –1616. Liu, X.J., Sorisky, A., Zhu, L., Pawson, T., 1995. Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation. Mol. Cell Biol. 15, 3563 –3570. Lutz, L.B., Kim, B., Jahani, D., Hammes, S.R., 2000. G protein beta gamma subunits inhibit nongenomic progesterone-induced signaling and maturation in Xenopus lae6is oocytes. Evidence for a release of inhibition mechanism for cell cycle progression. J. Biol. Chem. 275, 41512 – 41520. Maller, J.L., 2001. The elusive progesterone receptor in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 98, 8 – 10. Maller, J.L., Krebs, E.G., 1977. Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3%:5%-monophosphatedependent protein kinase. J. Biol. Chem. 252, 1712 –1718. Maller, J.L., Krebs, E.G., 1980. Regulation of oocyte maturation. Curr. Top. Cell. Regul. 16, 271 – 311. Masui, Y., Market, C.L., 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129 – 146. Mitra, J., Schultz, R.M., 1996. Regulation of the acquisition of meiotic competence in the mouse: changes in the subcellular localization of cdc2, cyclin B1, cdc25C and wee1, and in the concentration of these proteins and their transcripts. J. Cell Sci. 109, 2407 – 2415. Moor, R.M., Smith, M.W., Dawson, R.M.C., 1980. Measurement of intercellular coupling between oocytes and cumulus cells using intracellular markers. Exp. Cell Res. 126, 15 –29. Moore, G.D., Ayabe, T., Kopf, G.S., Schultz, R.M., 1996. Temporal patterns of gene expression of G1-S cyclins and cdks during the first and second mitotic cell cycles in mouse embryos. Mol. Reprod. Dev. 45, 264 –275. Muslin, A.J., Klippel, A., Williams, L.T., 1993. Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation. Mol. Cell. Biol. 13, 6661 –6666. Pincus, G., Enzmann, E.V., 1935. The comparative behavior of mammalian eggs in vivo and in vitro: I. The activation of ovarian eggs. J. Exp. Med. 62, 665 –675. Richards, J.S., 1980. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol. Rev. 60, 51 –89.
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Richard, F.J., Tsafriri, A., Contil, M., 2001. Role of phosphodiesterase type 3A in mammalian oocyte maturation, Biol Reprod., in press. Sagata, N., Watanabe, N., Vande Woude, G.F., Ikawa, Y., 1989. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 342, 512 – 518. Schultz, R.M., Montgomery, R.R., Belanoff, J.R., 1983. Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev. Biol. 97, 264 – 273. Sheng, Y., Tiberi, M., Booth, R.A., Ma, C., Liu, X.J., 2001. Regulation of Xenopus oocyte meiosis arrest by G protein betagamma subunits. Curr. Biol. 11, 405 – 416. Shitsukawa, K., Andersen, C.B., Richard, F.J., Horner, A.K., Wiersma, A., van Duin, M., Conti, M., 2001. Cloning and characterization of the cGMP-inhibited phosphodiesterase PDE3A expressed in mouse oocyte. Biol. Reprod. 65, 188 – 196. Simon, A.M., Goodenough, D.A., Li, E., Paul, D.L., 1997. Female infertility in mice lacking connexin 37. Nature 385, 525 –529. Simoncini, T., Hafezi-Moghadam, A., Brazil, D.P., Ley, K., Chin, W.W., Liao, J.K., 2000. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407, 538 – 541. Taieb, F., Thibier, C., Jessus, C., 1997. On cyclins, oocytes, and eggs. Mol. Reprod. Dev. 48, 397 – 411. Tian, J., Kim, S., Heilig, E., Ruderman, J.V., 2000. Identification of XPR-1, a progesterone receptor required for Xenopus oocyte activation. Proc. Natl. Acad. Sci. USA 97, 14358 – 14363. Tsafriri, A., Channing, C.P., 1975. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology 96, 922 – 927. Tsafriri, A., Dekel, N., 1994. Molecular mechanisms in ovulation. In: Findlay, J.K. (Ed.), Molecular Biology Female Reproductive System. Academic Press, San Diego, pp. 207 – 258. Tsafriri, A., Chun, S.Y., Zhang, R., Hsueh, A.J.W., Conti, M., 1996. Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev. Biol. 178, 393 – 402. Tsafriri, A., Pomerantz, S.H., Channing, C.P., 1976. Inhibition of oocyte maturation by porcine follicular fluid: partial characterization of the inhibitor. Biol. Reprod. 14, 511 – 516. Vivarelli, E., Conti, M., De Felici, M., Siracusa, G., 1983. Meiotic resumption and intracellular cAMP levels in mouse oocytes treated with compounds which act on cAMP metabolism. Cell Differ. 12, 271 – 276. Wiersma, A., Hirsch, B., Tsafriri, A., Hanssen, R.G., Van de Kant, M., Kloosterboer, H.J., Conti, M., Hsueh, A.J., 1998. Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents. J. Clin. Invest. 102, 532 – 537.
Role of cyclic nucleotide signaling in oocyte maturation Marco Conti a,*, Carsten Bo Andersen a, Francois Richard b, Celine Mehats a, Sang-Young Chun c, Kathleen Horner a, Catherine Jin a, Alex Tsafriri d a
Di6ision of Reproducti6e Biology, Department of Gynecology and Obstetrics, Stanford Uni6ersity School of Medicine, Stanford, CA 94305, USA b Centre de Recherche en Biologie de la Reproduction, De´partement des sciences animals, Uni6ersite´ La6al, Ste-Foy, Que., Canada c Hormone Research Center, Chonnam National Uni6ersity, Kwangju, South Korea d The Weizmann Institute of Science, Department of Biological Regulation, Reho6ot 76100, Israel
Abstract 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. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Oocyte maturation; Meiosis; cAMP; Mammalian; Xenopus
1. Meiosis and meiotic competence of the mammalian oocyte Unlike male germ cells that enter meiosis only at puberty, the ontogeny of the mammalian oocyte is characterized by two consecutive phases of meiotic progression/arrest that span the embryonic, fetal, pubertal, and adult life. In most mammalian species, the first meiotic division is initiated during prenatal life or shortly after birth (Eppig, 1993; Tsafriri and Dekel, 1994). Once the transition between prophase and
* Corresponding author. Tel.: + 1-650-725-2452; fax: +1-650-7257102. E-mail address: [email protected] (M. Conti).
metaphase (diplotene stage) is reached (around birth), unknown mechanisms produce a meiotic arrest in the late prophase that is maintained until shortly before ovulation. Rather than continuing in their condensation, chromosomes disperse in the nucleus, and transcription is resumed. In the growing preantral follicle, oocytes remain in this diplotene stage and are incompetent to resume meiosis. This inability to re-enter the cell cycle is probably due to absence, or accumulation below a threshold, of key components necessary for the activation of the maturation promoting factor (MPF) (Kanatsu-Shinohara et al., 2000; Mitra and Schultz, 1996; Moore et al., 1996). With growth of the oocyte in the preovulatory follicle, the components involved in the control of the cell cycle accumulate to a level permissive for meiosis (meiotic competence). Meiotic
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resumption of the oocyte, however, is prevented by inhibitory signals derived from the somatic compartment. In rat, the oocyte will resume meiosis 2– 3 h after the preovulatory gonadotropin surge. The prominent nucleus, characteristic of the dictyate oocyte (germinal vesicle, GV), disappears following chromosome condensation and dissolution of the nuclear membrane GV breakdown (GVBD). Meiosis continues with chromosome segregation, asymmetric division, and extrusion of a polar body, and, unlike mitosis, it proceeds without an interphase and DNA replication to a second division, which is arrested in metaphase II (second meiotic arrest). A cytostatic factor (CSF) (Masui and Market, 1971), of which the mos kinase is an essential component (Sagata et al., 1989), is responsible for maintaining the metaphase II arrest. Only a metaphase II-arrested secondary oocyte is fertilizable by spermatozoa.
2. The follicle environment maintains the oocyte maturational arrest In all mammalian species examined, oocytes that are removed from the follicle undergo spontaneous maturation (GVBD), suggesting that the follicular environment prevents the maturation of the oocyte (Pincus and Enzmann, 1935; Edwards, 1965). In some instances, it has been shown that cumulus oocyte complexes need to physically interact with mural granulosa cells to maintain meiotic arrest (Hillensjo¨ et al., 1979), indicating that intercellular communications between the cells of the follicle, perhaps mediated by gap junctions, are indispensable for maintaining this arrest. In other cases, this physical communication between somatic cells and the oocyte was not necessary because follicular fluid inhibited resumption of meiosis (Tsafriri and Dekel, 1994). These observations have led to the hypothesis that an oocyte meiotic inhibitor (often termed OMI) is produced by granulosa cells. Several candidates have been proposed for this inhibitory activity. Tsafriri et al. have proposed that a peptide of less than 2000 Da is responsible for inhibition of meiosis (Tsafriri and Channing, 1975; Tsafriri et al., 1976; Tsafriri and Dekel, 1994); however, the biochemical nature of this protein remains unknown to date. Several studies indicate that hypoxanthine may fulfill the properties of this inhibitor of oocyte maturation (Eppig et al., 1985; Eppig and Downs, 1987; Downs et al., 1989). Hypoxanthine is present in the follicular fluid at 1– 2 mM, a concentration sufficient to inhibit oocyte maturation (Eppig et al., 1985). Finally, it is possible that cAMP, either produced locally or transferred to the oocyte through gap junctions, may function as the inhibitory signal produced by granulosa cells (Dekel, 1988; see below).
3. The blockade of meiotic resumption is due to elevated cAMP levels in the oocyte Studies in species as diverse as starfish, Xenopus, and mammals have reached the identical conclusion that levels of the second messenger cAMP in the oocyte play a critical role in maintaining meiotic arrest (Ferrell, 1999; Taieb et al., 1997). High cAMP levels in the oocyte maintain protein kinase A (PKA) in an active/ dissociated state which causes phosphorylation of unknown protein substrates (Fig. 1). Classic studies using injections of the PKA inhibitor, PKI, or regulatory and catalytic subunits of PKA in Xenopus oocytes, support this model (Maller and Krebs, 1980, 1977). Similar findings with injections of PKA have been reported for mouse oocytes (Bornslaeger et al., 1986), and pharmacological manipulations to increase cAMP levels in mammalian oocytes invariably produces a blockade of meiosis (reviewed in Conti et al., 1998). Consistent with this hypothesis, it has long been known that phosphodiesterase (PDE) inhibitors, such as the xanthine IBMX, block spontaneous mammalian oocyte maturation (Conti et al., 1998), but the exact PDE target for this pharmacological intervention was unknown until recently (see below). In mammalian oocytes of the preovulatory follicle, how cAMP is maintained above a threshold is still unclear (Fig. 1). Conflicting results have been reported on the expression of the enzyme adenylyl cyclase (AC) responsible for cAMP synthesis, and it is uncertain whether the oocyte is able to produce sufficient cAMP to prevent meiosis (Schultz et al., 1983; Dekel and
Fig. 1. cAMP homeostasis and the meiotic arrest. See details in the test.
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Beers, 1980; Tsafriri and Dekel, 1994). As an alternative hypothesis, it has been proposed that cAMP diffuses from cumulus cells to the oocyte through gap junctions (Dekel, 1988; Eppig, 1991) (Fig. 1). This diffusion, perhaps in synergy with the inhibition of the oocyte PDE by hypoxanthine, maintains cAMP levels high enough to prevent meiotic resumption. Although gap junctions play a critical role in oocyte/cumulus cell communication, as demonstrated by the inactivation of connexin 37 (Simon et al., 1997) by homologous recombination, this latter knockout model does not provide information on the role of gap junctions at the time of meiotic resumption (Carabatsos et al., 2000).
4. PDE3A as an essential component of the signaling pathway that controls the meiotic arrest Our laboratory has identified the major PDE form expressed in rat and mouse oocyte as a PDE3A and found that specific inhibitors for this class of PDEs prevents oocyte maturation in vitro and in vivo (Tsafriri et al., 1996; Wiersma et al., 1998), therefore demonstrating an essential role of this enzyme in meiotic arrest (Fig. 1). The presence of PDE3A in rat and mouse oocytes has been confirmed by biochemical and molecular approaches. Complementary DNAs containing a complete PDE3A open reading frame were retrieved from a mouse oocyte library (Shitsukawa et al., 2001). Expression of the oocyte PDE cDNA was associated with PDE activity with the properties of a PDE3A described in human. Western blot analysis of the recombinant protein demonstrated the expression of a polypeptide of approximately 130 kDa. Polyclonal antibodies have been produced by injecting a GST fused to the carboxyl terminus of the oocyte PDE3A. The recombinant PDE3A is recognized by this antibody (Shitsukawa et al., 2001). The same antibody immunoprecipitates 90% of the PDE activity from mouse or rat oocytes in a specific manner, confirming that a PDE3A is the major PDE expressed in oocytes. Preliminary evidence suggests that a splicing variant of PDE3A, devoid of the membrane anchoring domain, may be expressed in oocytes (Shitsukawa et al., 2001). Taken together, these studies provide biochemical and immunological evidence that PDE3A is expressed and is active in rodent oocytes. Preliminary data obtained with Rhesus macaque oocytes further indicate that PDE3s play an important role in meiotic arrest in primates as well (Jensen et al., 2000). Detailed pharmacological profiling studies to complement the characterization of the PDE3 expressed in rodent oocytes demonstrate that PDE3A controls a pool of cAMP critical for regulation of meiotic resumption (Shitsukawa et al., 2001). Selective and nonselec-
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tive PDE inhibitors were used at a range of concentrations between 10 − 10 and 10 − 3 M in PDE assays using recombinant PDE3A cloned from the oocytes, and PDE from extracts of cumulus enclosed oocytes (COCs). The rank of potency was then compared to the potency of the compounds in inhibiting resumption of meiosis (measured as GVBD). This comparison demonstrated a highly statistically significant correlation between the potency of the compounds in inhibiting recombinant PDE3A, PDE derived from the oocytes, and GVBD. These findings provide further evidence that the PDE that controls the cAMP pool involved in meiosis resumption is a PDE3A. It should be noted that hypoxanthine, which is thought to be a natural inhibitor of meiotic resumption, inhibited the oocyte PDE3A with a potency very similar to that measured for inhibition of meiotic resumption. This in vitro observation confirms and extends in vivo experiments injecting PDE3-specific inhibitors in rat and mice (Wiersma et al., 1998). These inhibitors caused a dissociation between meiotic maturation and ovulation. As a result, ovulated oocytes from PDE3treated rats or mice were still in a GV state and could not be fertilized. Because granulosa cells were not affected by this treatment, this observation provides ‘proof of concept’ for targeting oocyte maturation as an effective method of fertility control (Wiersma et al., 1998). Collectively, the above reviewed data demonstrate that PDE3A plays an essential role in the maintenance in meiotic arrest of the oocyte not only in vitro, but also in vivo. Our findings also show that signals for resumption of meiosis require an active PDE3A.
5. How is meiosis initiated? During the normal ovulatory cycle, the physiological stimulus for meiotic resumption is the LH surge (Tsafriri and Dekel, 1994). Pharmacological blockade of the LH surge with Nembutal, GnRH antagonists, or hypophysectomy prevents oocyte maturation. Similarly, inactivation of the LH receptor is associated with a failure of the oocyte to undergo meiotic resumption and ovulation. Because gonadotropin receptors are not detectable in the oocyte, it has been concluded that LH stimulates granulosa cells and that this stimulation removes the inhibitory constraint on meiotic resumption, perhaps by blocking the production of an inhibitory substance (Fig. 2). Several studies have indicated that oocyte maturation is associated with a decrease in cAMP in both rat (Aberdam et al., 1987) and mouse (Vivarelli et al., 1983; Schultz et al., 1983) oocytes, suggesting that LH signals this decrease in cAMP. These findings are somewhat paradoxical because it is well established that LH pro-
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Fig. 2. Possible mechanisms involved in resumption of meiosis in mammals.
duces a large increase in cAMP in granulosa cells (Richards, 1980; Hsueh et al., 1984). To reconcile these discrepancies, it should be noted that, in spite of the presence of gap junctions, granulosa cells and oocytes act as two separate compartments in terms of cAMP action and an increase in cAMP in the somatic cell compartment is somehow translated into a decrease in cAMP in the oocyte. Such a situation is strongly suggested by our finding that manipulations of cAMP in the two compartments through selective inhibition of PDEs in somatic cells and oocytes produce opposing effects on maturation. Inhibitors of the oocyte PDE3 invariably block oocyte maturation induced by gonadotropins, whereas inhibition of PDE4 in granulosa cells produces meiotic resumption and ovulation in the absence of LH (Tsafriri et al., 1996). Studies with selective PKA inhibitors also are consistent with this hypothesis because activation of the PKA present in granulosa cells stimulates resumption of meiosis, whereas stimulation of the PKA in the oocytes blocks oocyte maturation (Downs and Hunzicker-Dunn, 1995). Gonadotropin-induced severance of the communication between cumulus cells and oocytes may be the trigger for a decrease in cAMP and resumption of meiosis. However, conflicting results have been published on this issue, with several studies indicating that a loss of communication via gap junctions lags behind resumption of meiosis by several hours (Moor et al., 1980; Dekel et al., 1981; Eppig, 1982). The above two possibilities are not mutually exclusive, as closure of gap junctions may cooperate with an active signal, which together decreases cAMP. Some of the data thus far published are also compatible with a more complex model whereby cumulus cells send a positive signal that activates the oocyte, overcoming the inhibitory cAMP levels (Fig. 2). This latter scenario is suggested by findings with an in vitro mouse model that is often used to study oocyte maturation
(Eppig et al., 1983; Freter and Schultz, 1984; Byskov et al., 1997). In this experimental paradigm, cumulus oocyte complexes (COC) are maintained in culture in the presence of hypoxanthine or dbcAMP, and meiotic resumption is induced by FSH, thus demonstrating that cumulus cells can send a positive stimulus to the oocyte that overrides the cAMP inhibitory effects. Stimuli other than the gonadotropin FSH, including EGF, GH, and TNFa, produce a similar effect, probably by acting on cumulus cells (Dekel and Sherizly, 1985; Apa et al., 1994; Feng et al., 1988). However, this stimulation is ineffective with PDE inhibitors more potent and specific than hypoxanthine, or high dbcAMP concentrations. It is, therefore, not clear whether this positive stimulus is effective in the presence of high cAMP levels or whether a decrease in cAMP is obligatory for the resumption of meiosis. Further studies under conditions where only the oocyte cAMP is affected by PDE3-specific inhibitors should provide further insight into the mechanisms underlying this ‘hormone-stimulated’ oocyte maturation. The quest for a positive signal originating from granulosa cells and inducing oocyte maturation has led to the isolation of MAS, a cholesterol derivative found in both testis and ovary (Byskov et al., 1995). FF-MAS is found in the follicular fluid, its concentration is increased by gonadotropins, and in vitro, FF-MAS induces resumption of meiosis of oocytes that are maintained in an arrested state by hypoxanthine (Byskov et al., 1997). Interestingly, differences have been found in the spontaneous and MAS-induced oocyte maturation, and it appears that MAS may be able to overcome the blockade imposed by high cAMP levels. Measurement of PDE present in the oocyte at different times during spontaneous maturation showed a transient increase in activity prior to GVBD (Richard et al., 2001). This increase was blocked by PDE3 inhibitors, but not PDE4, indicating that the oocyte PDE3 is involved in this regulation. Measurement of the PDE activity in oocyte derived from intact follicles incubated in vitro for 2 h with hCG also showed an increase in PDE3 activity. Thus, an increase in oocyte PDE activity precedes both spontaneous and hCG-induced oocyte maturation. These findings support the concept that an active signal is responsible for oocyte maturation and that an increase in PDE activity may be a component of the signaling cascade involved in re-entry into the cell cycle.
6. Which signal transduction pathway of the oocyte triggers GVBD and resumption of meiosis? The above findings are compatible with the hypothesis that oocyte maturation is dependent on the in-
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traoocyte cAMP levels and several signaling pathways are activated in the oocyte to either decrease cAMP levels or to override the cAMP inhibitory effects. The comparison with the Xenopus oocyte may provide important clues on the mechanism of meiotic resumption in mammalian oocytes. Progesterone produced by follicular cells is considered the physiological stimulus for resumption of meiosis in Xenopus oocytes (Maller and Krebs, 1980). Because IGF-1 potently activates resumption of meiosis in the frog, it is possible that the two signals cooperate under physiological conditions. Progesterone acts through a ‘nonnuclear’ steroid receptor to decrease cAMP synthesis by inhibiting adenylyl cyclase, but with unclear effects on PDE activity (Maller and Krebs, 1980; Ferrell, 1999). The regulation of the adenylyl cyclase by progesterone occurs by a mechanism different from the classical Gi-mediated inhibition and may depend on the level of free Gbg (Lutz et al., 2000; Sheng et al., 2001). On the contrary, a PDE activation via a PI3K/PKB regulation plays an essential role in IGF-1 signaling for re-entry into the cell cycle (Andersen et al., 1998; Liu et al., 1995; Muslin et al., 1993). Thus, both stimuli appear to converge in the Xenopus oocytes to produce a decrease in cAMP which, in turn, induces oocyte maturation. Although earlier work suggested the presence of a progesterone receptor exposed on the cell surface, recent data are consistent with the view that the ‘classical’ nuclear progesterone receptor is mediating the steroid effects on meiotic resumption in the Xenopus oocytes (Tian et al., 2000; Bayaa et al., 2000; Maller, 2001). Expression of an active mouse oocytes PDE3A in Xenopus oocytes produced GVBD to the same extent as progesterone or insulin (Andersen et al., manuscript in preparation). It also induced mos translation and ERK phosphorylation/activation, demonstrating that the complete program involved in the re-entry into the cell cycle is inducible by an increase in PDE activity. Moreover, a concentration response study demonstrated that a small increase in PDE activity was sufficient to produce meiotic resumption. Thus, an increase in PDE activity is sufficient to induce the complete pattern of changes that precede GVBD and re-entry into the cell cycle. Several findings, including our own, suggest that the progesterone and IGF-1 signaling pathways may be more integrated than originally thought. Inhibition of PI-3 kinase has some effects on the progesterone stimulation of resumption of meiosis (Muslin et al., 1993), and inhibition of PKB or PDE also interferes with progesterone activation (Andersen et al., manuscript in preparation). Interestingly, it has been recently proposed that steroid receptors are in a complex with PI-3 kinase and occupancy by steroid hormones causes acti-
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vation of this cascade (Simoncini et al., 2000; Maller, 2001), further implicating this pathway in the nongenomic action of steroids and in the control of meiotic maturation. The recent discovery that nuclear progesterone receptors are expressed in Xenopus oocytes is consistent with this view (Bayaa et al., 2000; Tian et al., 2000). Because IGF-1 stimulates resumption of meiosis in oocytes from the Xenopus lae6is via a PI3-K activation, we tested the hypothesis that the serine/threonine kinase PKB/Akt is involved in this process and that a PDE3 activation is distal to this kinase (Fig. 3). The activation of the PKB/Akt is thought to be a critical step in the phosphoinositide 3-OH kinase (PI3-K) pathway that regulates cell growth and differentiation. Injection of mRNA coding for a constitutively active PKB/Akt in Xenopus oocytes induced GVBD to the same extent as progesterone or insulin treatment (Andersen et al., 1998). The induction of GVBD was associated with the phosphorylation of the MAP kinase ERK2, indicating that all the components of the maturation process are activated in the injected oocytes. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a K179M mutation in the catalytic domain that abolishes the kinase activity, had no effect (Andersen et al., 1998). A kinase with a mutation in the myristoylation signal sequence did not induce GVBD, in spite of the high levels of expression. Thus, both the membrane interaction and the kinase activity of PKB/Akt are required to promote resumption of meiosis. The induction of GVBD by insulin/IGF-1 or PKB/Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE3 is required for the Akt action. More importantly, the constitutively
Fig. 3. Putative signaling pathways in regulation of meiosis in Xenopus oocyte.
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active Akt caused a two-fold increase in the PDE activity endogenous to the oocyte (Andersen et al., 1998). Finally, an antibody that blocks the Akt activity also blocks oocyte maturation induced by insulin. These data demonstrate that PKB/Akt is part of the mechanism controlling resumption of meiosis in the Xenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation. Given that PDE3 activity is essential for oocyte maturation in rodents, the comparison with Xenopus suggests that a PI3K/Akt/PDE3A pathway may be an important component in regulating meiotic resumption in mammals as well. In summary, although the exact intracellular signals controlling meiotic resumption in mammalian oocytes remains to be elucidated, homologies with the Xenopus model suggest that multiple pathways may participate in this control. These signaling pathways may converge in the regulation of cAMP levels, perhaps by regulating the PDE3 expressed in the oocytes, thus removing an inhibitory constraint. This decrease in cAMP may be sufficient to trigger meiotic resumption, but it is also possible that signals that override the cAMP inhibitory effect may participate in the control of oocyte maturation. Understanding these mechanisms of control will open new avenues for the pharmacological manipulation of fertility.
Acknowledgements The work from the authors’ laboratory was supported by NIH grant HD20788 and by NICHD/NIH through a cooperative agreement (U54 HD31398) as part of the Specialized Cooperative Centers Program in Reproduction Research.
References Aberdam, E., Hanski, E., Dekel, N., 1987. Maintenance of meiotic arrest in isolated rat oocytes by the invasive adenylate cyclase of Bordetella pertussis. Biol. Reprod. 36, 530 –535. Andersen, C.B., Roth, R.A., Conti, M., 1998. Protein kinase B/Akt induces resumption of meiosis in Xenopus oocytes. J. Biol. Chem. 273, 18705 – 18708. Apa, R., Lanzone, A., Miceli, F., Mastrandrea, M., Caruso, A., Mancuso, S., Canipari, R., 1994. Growth hormone induces in vitro maturation of follicle- and cumulus-enclosed rat oocytes. Mol. Cell. Endocrinol. 106, 207 –212. Bayaa, M., Booth, R.A., Sheng, Y., Liu, X.J., 2000. The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism. Proc. Natl. Acad. Sci. USA 97, 12607 – 12612. Bornslaeger, E.A., Mattei, P.M., Schultz, R.M., 1986. Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev. Biol. 114, 453 – 462. Byskov, A.G., Andersen, C.Y., Nordholm, L., Thogersen, H., Guoliang, X., Wassmann, O., Andersen, J.V., Guddal, E., Roed, T.,
1995. Chemical structure of sterols that activate oocyte meiosis. Nature 374, 559 – 562. Byskov, A.G., Andersen, C.Y., Hossaini, A., Guoliang, X., 1997. Cumulus cells of oocyte – cumulus complexes secrete a meiosis-activating substance when stimulated with FSH. Mol. Reprod. Dev. 46, 296 – 305. Carabatsos, M.J., Sellitto, C., Goodenough, D.A., Albertini, D.F., 2000. Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Dev. Biol. 226, 167 – 179. Conti, M., Andersen, C.B., Richard, F.J., Shitsukawa, K., Tsafriri, A., 1998. Role of cyclic nucleotide phosphodiesterases in resumption of meiosis. Mol. Cell. Endocrinol. 145, 9 – 14. Dekel, N., Regulation of oocyte maturation: the role of cAMP. In: In Vitro Fertilization and other Assisted Reproduction. Acad. Sci., New York, 1988, pp. 211 – 216. Dekel, N., Beers, W.H., 1980. Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus. Dev. Biol. 75, 247 – 254. Dekel, N., Sherizly, I., 1985. Epidermal growth factor induces maturation of rat follicle-enclosed oocytes. Endocrinology 116, 406 – 409. Dekel, N., Lawrence, T.S., Gilula, N.B., Beers, W.H., 1981. Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev. Biol. 86, 356 – 362. Downs, S.M., Daniel, S.A.J., Bornslaeger, E.A., Hoppe, P.C., Eppig, J.J., 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., Hunzicker-Dunn, M., 1995. Differential regulation of oocyte maturation and cumulus expansion in the mouse oocyte – cumulus cell complex by site-selective analogs of cyclic adenosine monophosphate. Dev. Biol. 172, 72 – 85. Edwards, R.G., 1965. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208, 349 –351. Eppig, J.J., 1982. The relationship between cumulus cell –oocyte coupling, oocyte meiotic maturation, and cumulus expansion. Dev. Biol. 89, 268 – 272. Eppig, J.J., 1991. Intercommunication between mammalian oocytes and companion somatic cells. Bioessays 13, 569 – 574. Eppig, J.J., 1993. Regulation of mammalian oocyte maturation. In: Adashi, E.Y., Leung, P.C.K. (Eds.), The Ovary. Raven Press, New York, pp. 185 – 208. Eppig, J.J., Downs, S.M., 1987. The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation. Dev. Biol. 119, 313 – 321. Eppig, J.J., Freter, R.R., Ward-Bailey, P.F., Schultz, R.M., 1983. Inhibition of oocyte maturation in the mouse: participation of cAMP, steroid hormones and a putative maturation-inhibitory factor. Dev. Biol. 100, 39 – 49. Eppig, J.J., Ward-Bailey, P.F., Coleman, D.L., 1985. Hypoxanthine and adenosine in murine ovarian follicular fluid: concentrations and activity in maintaining oocyte meiotic arrest. Biol. Reprod. 33, 1041 – 1049. Feng, P., Catt, K.J., Knecht, M., 1988. Transforming growth factorbeta stimulates meiotic maturation of the rat oocyte. Endocrinology 122, 181 – 186. Ferrell, J.E. Jr., 1999. Xenopus oocyte maturation: new lessons from a good egg. Bioessays 21, 833 – 842. Freter, R.R., Schultz, R.M., 1984. Regulation of murine oocyte meiosis: evidence for a gonadotropin-induced, cAMP-dependant reduction in a maturation inhibitor. J. Cell Biol. 98, 1119 –1128. Hillensjo¨ , T., Channing, C.P., Pomerantz, S.H., Schwartz-Kripner, A., 1979. Intrafollicular control of oocyte maturation in the pig. In Vitro 15, 32 – 39.
M. Conti et al. / Molecular and Cellular Endocrinology 187 (2002) 153–159 Hsueh, A.J., Adashi, E.Y., Jones, P.B., Welsh, T.H. Jr., 1984. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5, 76 – 127. Jensen, J., Schwinof, K., Hazzard, T., Zelinski-Wooten, M., Conti, M., Stouffer, R., 2000. In vitro exposure to phosphodiesterase 3 inhibitors prevents spontaneous resumption of meiosis in Rhesus macaque oocytes. In: Annual Meeting of the Society for the Study of Reproduction, 272pp. Kanatsu-Shinohara, M., Schultz, R.M., Kopf, G.S., 2000. Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes. Biol. Reprod. 63, 1610 –1616. Liu, X.J., Sorisky, A., Zhu, L., Pawson, T., 1995. Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation. Mol. Cell Biol. 15, 3563 –3570. Lutz, L.B., Kim, B., Jahani, D., Hammes, S.R., 2000. G protein beta gamma subunits inhibit nongenomic progesterone-induced signaling and maturation in Xenopus lae6is oocytes. Evidence for a release of inhibition mechanism for cell cycle progression. J. Biol. Chem. 275, 41512 – 41520. Maller, J.L., 2001. The elusive progesterone receptor in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 98, 8 – 10. Maller, J.L., Krebs, E.G., 1977. Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3%:5%-monophosphatedependent protein kinase. J. Biol. Chem. 252, 1712 –1718. Maller, J.L., Krebs, E.G., 1980. Regulation of oocyte maturation. Curr. Top. Cell. Regul. 16, 271 – 311. Masui, Y., Market, C.L., 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129 – 146. Mitra, J., Schultz, R.M., 1996. Regulation of the acquisition of meiotic competence in the mouse: changes in the subcellular localization of cdc2, cyclin B1, cdc25C and wee1, and in the concentration of these proteins and their transcripts. J. Cell Sci. 109, 2407 – 2415. Moor, R.M., Smith, M.W., Dawson, R.M.C., 1980. Measurement of intercellular coupling between oocytes and cumulus cells using intracellular markers. Exp. Cell Res. 126, 15 –29. Moore, G.D., Ayabe, T., Kopf, G.S., Schultz, R.M., 1996. Temporal patterns of gene expression of G1-S cyclins and cdks during the first and second mitotic cell cycles in mouse embryos. Mol. Reprod. Dev. 45, 264 –275. Muslin, A.J., Klippel, A., Williams, L.T., 1993. Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation. Mol. Cell. Biol. 13, 6661 –6666. Pincus, G., Enzmann, E.V., 1935. The comparative behavior of mammalian eggs in vivo and in vitro: I. The activation of ovarian eggs. J. Exp. Med. 62, 665 –675. Richards, J.S., 1980. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol. Rev. 60, 51 –89.
159
Richard, F.J., Tsafriri, A., Contil, M., 2001. Role of phosphodiesterase type 3A in mammalian oocyte maturation, Biol Reprod., in press. Sagata, N., Watanabe, N., Vande Woude, G.F., Ikawa, Y., 1989. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 342, 512 – 518. Schultz, R.M., Montgomery, R.R., Belanoff, J.R., 1983. Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev. Biol. 97, 264 – 273. Sheng, Y., Tiberi, M., Booth, R.A., Ma, C., Liu, X.J., 2001. Regulation of Xenopus oocyte meiosis arrest by G protein betagamma subunits. Curr. Biol. 11, 405 – 416. Shitsukawa, K., Andersen, C.B., Richard, F.J., Horner, A.K., Wiersma, A., van Duin, M., Conti, M., 2001. Cloning and characterization of the cGMP-inhibited phosphodiesterase PDE3A expressed in mouse oocyte. Biol. Reprod. 65, 188 – 196. Simon, A.M., Goodenough, D.A., Li, E., Paul, D.L., 1997. Female infertility in mice lacking connexin 37. Nature 385, 525 –529. Simoncini, T., Hafezi-Moghadam, A., Brazil, D.P., Ley, K., Chin, W.W., Liao, J.K., 2000. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407, 538 – 541. Taieb, F., Thibier, C., Jessus, C., 1997. On cyclins, oocytes, and eggs. Mol. Reprod. Dev. 48, 397 – 411. Tian, J., Kim, S., Heilig, E., Ruderman, J.V., 2000. Identification of XPR-1, a progesterone receptor required for Xenopus oocyte activation. Proc. Natl. Acad. Sci. USA 97, 14358 – 14363. Tsafriri, A., Channing, C.P., 1975. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology 96, 922 – 927. Tsafriri, A., Dekel, N., 1994. Molecular mechanisms in ovulation. In: Findlay, J.K. (Ed.), Molecular Biology Female Reproductive System. Academic Press, San Diego, pp. 207 – 258. Tsafriri, A., Chun, S.Y., Zhang, R., Hsueh, A.J.W., Conti, M., 1996. Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev. Biol. 178, 393 – 402. Tsafriri, A., Pomerantz, S.H., Channing, C.P., 1976. Inhibition of oocyte maturation by porcine follicular fluid: partial characterization of the inhibitor. Biol. Reprod. 14, 511 – 516. Vivarelli, E., Conti, M., De Felici, M., Siracusa, G., 1983. Meiotic resumption and intracellular cAMP levels in mouse oocytes treated with compounds which act on cAMP metabolism. Cell Differ. 12, 271 – 276. Wiersma, A., Hirsch, B., Tsafriri, A., Hanssen, R.G., Van de Kant, M., Kloosterboer, H.J., Conti, M., Hsueh, A.J., 1998. Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents. J. Clin. Invest. 102, 532 – 537.