/07/$15.00/0 Molecular Endocrinology 21(9): Copyright 2007 by The Endocrine Society doi: /me

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1 /07/$15.00/0 Molecular Endocrinology 21(9): Printed in U.S.A. Copyright 2007 by The Endocrine Society doi: /me MINIREVIEW Mechanisms Regulating Oocyte Meiotic Resumption: Roles of Mitogen-Activated Protein Kinase Cheng-Guang Liang, You-Qiang Su, Heng-Yu Fan, Heide Schatten, and Qing-Yuan Sun State Key Laboratory of Reproductive Biology, Institute of Zoology (C.-G.L., Q.-Y.S.) and Graduate School (C.-G.L.), Chinese Academy of Sciences, Beijing , China; The Jackson Laboratory (Y.-Q.S.), Bar Harbor, Maine 04609; Department of Molecular and Cellular Biology (H.-Y.F.), Baylor College of Medicine, Houston, Texas 77030; and Department of Veterinary Pathobiology (H.S.), University of Missouri-Columbia, Columbia, Missouri Oocyte meiotic maturation is one of the important physiological requirements for species survival. However, little is known about the detailed events occurring during this process. A number of studies have demonstrated that MAPK plays a pivotal role in the regulation of meiotic cell cycle progression in oocytes, but controversial findings have been reported in both lower vertebrates and mammals. In this review, we summarized the roles of MAPK cascade and related signal pathways in oocyte OVERVIEW OF OOCYTE MEIOTIC RESUMPTION IN THE ANIMAL kingdom, oocyte maturation plays a crucial role for species survival, but the mechanisms controlling oocyte maturation are somewhat different in lower vertebrates (such as Xenopus) compared with mammals (such as mouse). During Xenopus oogenesis, the follicle-enclosed oocyte is arrested at the diplotene stage of meiotic prophase. When the oocyte reaches full size, it becomes competent to respond to progesterone. These oocytes are termed fully grown oocytes. In vitro, Xenopus fully grown oocytes remain First Published Online May 29, 2007 Abbreviations: AR, Androgen receptor; AREG, amphiregulin; Cdc2, cell division cycle 2; Cdc25C, cell division cycle 25 homolog C; CEO, cumulus-enclosed oocyte; Cx, connexin; DO, denuded oocyte; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EREG, epiregulin; FGF, fibroblast growth factor; GRP, G protein-coupled receptor; GVBD, germinal vesicle breakdown; HX, hypoxanthine; MAS, meiosis activating sterol; MEK, MAPK kinase; MI, metaphase I; MII, metaphase II; mos-ko, mos-knockout; MPF, maturation promoting factor; Myt1, myelin transcription factor 1; OA, okadaic acid; PDE, phosphodiesterase; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C; Plx1, polo-like kinase 1. Molecular Endocrinology is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community. meiotic reinitiation in both lower vertebrates and mammals. We also tried to reconcile the paradoxical results and highlight the new findings concerning the function of MAPK in both oocytes and the surrounding follicular somatic cells. The unresolved questions and future research directions regarding the role of MAPK in meiotic resumption are addressed. (Molecular Endocrinology 21: , 2007) in meiotic arrest until stimulated by exogenous steroids or growth factors. Oocyte meiosis in most mammals is initiated during fetus development and is arrested at the diplotene stage of the first meiotic prophase around the time of birth. Oocytes are maintained at this stage for weeks and even years depending on the species. At puberty, the oocytes that have almost reached their full sizes acquire full competence to resume meiosis. In vivo, meiosis in fully grown oocytes is maintained by constitutively active G protein-coupled receptors (GPRs), particularly GPR3, which is localized in oocytes (1, 2) and by meiotic inhibitory factors that are produced by follicular somatic cells within antral follicles. The resumption of meiosis is induced by the surge of preovulatory LH (3, 4). In vitro, contrary to Xenopus oocytes, mammalian oocytes can resume meiosis spontaneously when released from antral follicles and cultured under suitable conditions. However, meiotic inhibitors, such as hypoxanthine (HX) (5, 6), camp analogs (7), or phosphodiesterase (PDE) inhibitors (8), can block the spontaneous maturation, whereas their inhibitory effects can be overcome by the administration of gonadotropins (9). Moreover, oocytes cultured in intact large antral follicles can also resume meiosis when treated with hormones and growth factors, such as LH (10), GnRH (11), epidermal growth factor (EGF) 2037

2 2038 Mol Endocrinol, September 2007, 21(9): Liang et al. Minireview (12), EGF-like growth factors (13), and Leydig insulinlike 3 (14). The resumption of meiosis is manifested by germinal vesicle breakdown (GVBD), which is followed by chromosome condensation and metaphase I (MI) spindle assembly, homologous chromosome segregation, and the completion of first meiotic division. Meiosis is arrested again at the metaphase II (MII) stage until fertilization. BALANCE BETWEEN KINASES AND PHOSPHATASES PLAYS AN IMPORTANT ROLE IN THE CONTROL OF OOCYTE MEIOTIC RESUMPTION Because the transcriptional process in oocyte itself is repressed during meiotic maturation, the proteins that are translated from the stocked maternal mrnas and protein phosphorylation or dephosphorylation regulated by a series of kinases and phosphatases guarantee the accurate response of the oocyte to external stimulation (15). In regard to the signal initiation, the exact receptor responses to the physiological steroid remain poorly understood in lower vertebrates (16). The immediate event downstream from steroid stimulation in oocytes is the inactivation of adenylate cyclase and subsequent decrease of camp. There is a general consensus that a transient decrease in the camp level in oocytes is an obligatory step in the induction of meiotic maturation. This, in turn, is thought to result in decreased protein kinase A (PKA) activity and lead to dephosphorylation of a putative maturation inhibiting phosphorylated protein (15). The other role of PKA inactivation in oocytes is to promote the synthesis of a small number of proteins necessary for meiotic maturation. MOS and cyclin B are the most potential candidates because they are synthesized de novo after PKA inactivation (17). After signal initiation stimulated by steroids, the induction events will be transduced from the membrane to the nuclei. Again, a large number of protein kinases and phosphatases have been proposed to participate in this process. One of the protein kinases that might directly or indirectly participate in the regulation of oocyte meiotic resumption is the MAPK, specifically MAPK3/1, also commonly known as ERK1/2. The activation and function of MAPK involves a cascade of protein phosphorylation. The upstream regulator of MAPK is the MAPK kinase, also called MAP2K or MEK, which phosphorylates ERK1/2 on both serine and threonine residues (18). MEK is also activated by phosphorylation, and its upstream kinase in vertebrate oocytes is MOS, the product of the proto-oncogene c-mos. MOS is a 39-kDa germ cell-specific Ser/Thr protein kinase that was first identified in cells transformed by Moloney murine sarcoma virus (19). One of the immediate downstream targets of MAPK is p90 rsk [also known as RPS6KA2 (ribosomal protein S6 kinase, 90kDa, polypeptide 2)], which is activated by ERK1/2 in vitro and in vivo via phosphorylation on Ser369 and Thr577 (20). Among the multiple molecules regulating oocyte maturation, the most important kinase is the maturation promoting factor (MPF), which performs a dominant role in GVBD. MPF is a heterodimer kinase composed of the regulatory subunit cyclin B1 and the catalytic subunit cell division cycle 2 (Cdc2) (also termed cyclin-dependent kinase 1 or p34 cdc2 ) (21). In G 2 -arrested oocytes, the widely distributed form of MPF is pre-mpf, which is maintained in its inactive form by phosphorylation of Cdc2 on Thr14 and Tyr15 (22). These inhibitory phosphorylations are probably catalyzed by the myelin transcription factor 1 (Myt1) protein kinase, whereas dephosphorylation of these residues requires the 25 homolog C [cell division cycle 25 homolog C (Cdc25C)] phosphatase. Thus, the activation of MPF may be brought about by the direct activation of Cdc2, the control of the balance between Cdc25C and Myt1 activities, or both. It should be noted that most of the Cdc2 stock in the oocyte is monomeric, and only about 10% of Cdc2 is associated with cyclin B in the pre-mpf complexes. Thus, MPF can also be activated through supplying cyclin B to Cdc2, which is already phosphorylated on Thr161. This provides a direct activation of MPF by a cyclin binding approach (23). In the past decade, remarkable progress has been made in clarifying the role of the MAPK cascade in the regulation of oocyte meiotic resumption and meiotic progression. Pioneering research on the role of MAPK cascade in the regulation of Xenopus oocyte meiosis has been well reviewed (24 28). The partial role of the MAPK cascade in regulating mammalian germ cell functions, especially oocyte maturation and fertilization, have also been reviewed by us (29, 30). In this review, we will summarize the recent progress in our laboratories and others regarding the role of MAPK cascade in regulating oocyte meiotic resumption, and we will highlight the potential role of MAPK-dependent pathways in follicular somatic cells in the induction of meiotic resumption. THE ROLE OF MAPK DURING OOCYTE MEIOTIC RESUMPTION IN LOWER VERTEBRATES The relationship between the MAPK pathway and meiotic resumption has been investigated initially in Xenopus oocytes. Because inconsistent results have been reported in each step from the onset triggered by hormone stimulation to the final steps resulting in GVBD, we will try to reconcile these results in a perspective of three successive phases: signal initiation concerning the relationship between hormone stimulation and MAPK activation, signal transmission concerning the multiform pathways related to MAPK phosphorylation, and signal effects concerning the in-

3 Liang et al. Minireview Mol Endocrinol, September 2007, 21(9): teraction between MAPK and MPF. The powerful signal feedback loop will also be evaluated. Here, we need to mention that, although existing evidence has indicated that Xenopus oocytes express only ERK2 and MEK1 (31 33), for simplicity, we will use the general name MAPK and MEK to refer to Xenopus ERK2 and MEK1, respectively. Hormone-Related Activation of MAPK during Oocyte Meiotic Resumption in Lower Vertebrates Up to now, several hormones have been used to study the meiotic mechanism in lower vertebrate oocytes, including the steroids progesterone and androgens (34 36). In addition, insulin and some growth factors such as IGF and fibroblast growth factor (FGF) have also been used for oocyte maturation induction in vitro (37, 38), although it is not clear whether these hormones play a role in vivo. Nevertheless, the most commonly accepted physiological hormone that induces GVBD in lower vertebrates is progesterone. MOS/MAPK pathway plays a major role in progesterone-induced meiotic resumption in Xenopus oocytes. In Xenopus, progesterone is synthesized and released by follicle cells in response to pituitary hormones. Several lines of evidence have suggested that MAPK activation in Xenopus oocytes is essential for progesterone-induced MPF activation and GVBD. For example, progesterone-induced GVBD is accompanied by the activation of MAPK in vivo (39). Microinjection of constitutively activated thiophosphorylated MAPK or MEK into Xenopus oocytes can mimic progesterone-induced MPF activation and GVBD (40, 41). Conversely, when MAPK phosphatase CL100 or anti-xenopus MEK antibody is microinjected into immature oocytes, progesterone-induced MAPK activation and subsequent H1 kinase activation and GVBD are prevented (42, 43). These results indicate that MAPK mediates progesterone function in Xenopus oocyte maturation. A number of studies have explained the mechanism of progesterone-induced MAPK activation. The most likely protein that is downstream of progesterone and activates MAPK is MOS. As mentioned above, MOS is one of the proteins that are synthesized after progesterone stimulation. Progesterone-induced mos mrnas polyadenylation and translation has been shown to be essential for meiotic resumption (44, 45). Microinjected MOS can trigger meiotic maturation in the absence of progesterone stimulation (46). Thus, when mos mrna is inhibited by microinjection of mos-specific antisense oligonucleotides into Xenopus oocytes, progesteroneinduced Cdc2 activation and GVBD is inhibited (47). As a maturation-inducing factor, the function of MOS is mainly based on the activation of MEK and subsequent MAPK. It thus has been hypothesized that progesterone induces synthesis of the proto-oncogene protein MOS, which activates the MAPK cascade via phosphorylation of MEK (48), followed by the MOS- MEK-MAPK cascade inducing MPF activation and subsequent GVBD (28, 49, 50). However, this plausible model encountered challenging results that clearly showed that progesteroneinduced GVBD does not require MOS in Xenopus oocytes (51). Indeed, as early as 1991, some reports indicated that Ras is an alternative kinase existing in Xenopus oocytes to activate Cdc2 through a MOSindependent pathway. Subsequent studies supporting this conclusion showed that injection of Xenopus oocytes with V12 H-Ras (52) or its downstream kinase Raf can trigger meiotic resumption without progesterone stimulation and independent of MOS (53, 54). Nevertheless, Ras-induced meiotic resumption still needs MAPK activation because Ras is able to activate MAPK and S6 kinase and thus leads to GVBD (53, 55), whereas dominant-negative forms of Raf can impair MAPK activation induced by Ras and progesterone in Xenopus oocytes (56 58). Ras may contribute to progesterone-induced maturation by interacting with phosphatidylinositol 3-kinase (PI3K) because PI3K-related enzyme is crucial for human Ras-induced MPF activation (59). In fact, PI3K can coprecipitate with the Xenopus classical progesterone receptor (60), and dissociation of PI3K with the platelet-derived growth factor receptor inhibits progesterone-induced maturation (61). In regard to the results above, it appears that both MOS and Ras participate in progesterone-induced meiotic resumption, because inhibition of any single pathway, MOS/MEK or PI3K/Ras/Raf, is not able to block GVBD induced by progesterone. In contrast, Xenopus oocyte maturation is effectively blocked by inhibiting MOS function in addition to abrogating PI3K activity (62). However, although the H-Ras/PI3K pathway is functional in Xenopus oocytes, its role in progesterone-induced oocyte maturation under physiological conditions is questionable (59). First, all of the experiments reported up to now in Xenopus oocytes have been performed using human H-Ras but not the inherent Xenopus K- and N-Ras (63 65). The kinetics of maturation induced by H-Ras is delayed by 12 h compared with that induced by progesterone. Thus, all features of the human Ras effects are different from the physiological pathway induced by progesterone. Second, H-Ras induces only a partial activation of MAPK when protein synthesis and MPF activation are prevented. Full MAPK activation is reached only when MPF is activated and MOS is present (52). Third, the role of PI3K has been reported to be essential for insulin- but not progesterone-stimulated resumption of meiosis, because the PI3K inhibitor wortmannin only delayed progesterone-induced maturation but completely blocked the insulin-dependent maturation (60). According to these studies, a more reasonable explanation is that, under physiological condition, MOS is still the pivotal kinase for progesterone-induced GVBD. Although injection of morpholino antisense oligonucleotides of MOS does not prevent meiotic resumption in response to progesterone, the effect resulting in GVBD is delayed for 2 h compared

4 2040 Mol Endocrinol, September 2007, 21(9): Liang et al. Minireview with progesterone-treated control oocytes. This suggests that MOS plays a major role in regulating the progression of normal progesterone-triggered oocyte maturation. Together, it appears that activation of MOS is still the most efficient way to induce meiosis resumption, although other pathways can be used when MOS pathway is prevented. MAPK is a potential mediator for androgen-induced meiotic resumption under physiological conditions. Although most early studies used progesterone to trigger oocyte maturation, recent studies doubt its physiological role as the meiotic initiator (66). The most recent evidence suggests that androgens (especially testosterone) are the primary physiological steroids produced by Xenopus laevis ovaries and may promote oocyte maturation in vivo through classical androgen receptors (ARs) expressed in oocytes (67 69). In fact, inhibition of androgen production markedly reduces oocyte maturation and delays gonadotropin-mediated ovulation (70). One recent report proposed that androgen has a higher potential to induce oocyte maturation than progesterone under physiological condition (66). The immediate downstream reaction of androgen-induced AR activation is the inhibition of constitutive G protein signaling (especially G ), which maintains oocytes in meiotic arrest (66). A possible kinase downstream from G is PI3K (71), which has already been shown to activate MAPK through Ras/Raf pathway, but this hypothesis needs additional investigation. The other direct evidence is that reduction of AR expression by RNA interference abrogates MAPK activation and oocyte maturation triggered by low concentrations of testosterone (67, 69, 70). These results suggest that classical AR mediates nongenomic signaling through the MAPK pathway (67, 69, 70). Nevertheless, the definitive identification and characterization of the physiological steroid and receptor as well as the role of MAPK in androgeninduced meiotic resumption in Xenopus oocyte remains an important goal for future studies. Insulin- and growth factor-induced meiotic resumption requires the activation of MAPK. Insulin and other growth factors such as IGF-I and FGF have also been reported to induce meiosis resumption through MAPK in Xenopus oocytes. Ras/Raf pathway plays an important role in mediating the function of these factors. For example, it has been shown that insulin and IGFs can trigger meiotic resumption through receptor tyrosine kinase and PI3K (58, 72), followed by activation of the MAPK pathway via the GTP-binding protein Ras (73, 74). Because prevention of MAPK activation delays meiotic resumption induced by insulin (75), it can be concluded that MAPK plays an important role in normal meiotic resumption induced by insulin and IGFs. FGF-stimulated MAPK activation may be mediated by Src/Ras-dependent pathway because inhibition of Src will prevent Ras activation and GVBD (76, 77). In addition, the Src homology 2 domain of Src and PP2 [4-amino-5-(4- chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine], an inhibitor of Src, can also abolish MAPK activation. Thus, the transduction cascade induced by FGF in Xenopus oocytes involves the Src/Ras/MAPK cascade (78, 79). However, it should be noted that, although Src can activate MAPK and thus participates in oocyte maturation induced by FGF (80), inhibition of Src has minimal effect on testosterone-induced oocyte maturation (81). It appears that Src can not be activated by steroid in oocytes and does not appear to be important for steroid-triggered maturation. These differences indicate that classical steroid receptorregulated G protein signaling is more important in oocytes than Src activation, which may predominate in somatic cells. MAPK Plays a Major Role in Transmitting Signals from Steroid Stimulation to MPF Activation The above information provides indications that most of the hormone-induced meiotic resumption needs the activation of MAPK in Xenopus oocytes. However, a number of reports clearly showed that, in some cases, MAPK-independent meiotic resumption does exist in Xenopus, Chaetopterus, marine worm, and goldfish oocytes, because inhibition of MAPK activity by overexpression of the MAPK phosphatase Pyst1 (dual specificity protein phosphatase), or by prevention of MEK through the pharmacological inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] or indirectly through geldanamycin, cannot block MPF activation and GVBD induced by steroids (82 88). One reasonable explanation is that MPF activation and subsequent GVBD can be induced through another pathway that does not require MAPK activation. A recent study showed that, in Xenopus oocytes, MOS may activate MPF by directly inactivating Myt1, a kinase that phosphorylates and inhibits Cdc2 (89). This mechanism can bypass the activation of MAPK and induce MPF activation as well as meiotic resumption in Xenopus oocytes. Similarly, H-Ras-induced MPF activation can also bypass MAPK activation (52). So far, Raf and PI3K have been well characterized as the effectors of Ras in oocytes. Raf can activate the MAPK pathway, whereas PI3K is implicated in phosphatidylinositol metabolism (90 92). In fact, PI3K may contribute to steroid-induced maturation by promoting the Akt (or protein kinase B)-dependent stimulation of type 3 camp PDE (93). Moreover, the PI3K/AKT pathway may induce oocyte GVBD by inhibition of Myt1 in a MAPK-independent manner, which consequently changes the balance of activity between Myt1 and Cdc25C and subsequently activates MPF (94). Collectively, these data indicate that GVBD is not fully dependent on MAPK activation because MPF can be activated by MAPK-independent mechanism(s). It should be pointed out, however, that, although inhibition of MAPK does not block GVBD in some cases, geldanamycin-treated oocytes mature with significantly reduced efficiency (86), and U0126 only al-

5 Liang et al. Minireview Mol Endocrinol, September 2007, 21(9): lows GVBD to occur in oocytes freshly dissected from primed Xenopus (87). Suppression of MAPK activation does not affect the formation or activation of Cdc2- cyclin B complexes but reduces the level of active Cdc2 kinase and delays the occurrence of GVBD (86). Furthermore, although U0126-treated oocytes could have MPF activation and undergo GVBD during steroid treatment, they fail to form MI spindles, reaccumulate cyclin B, and hyperphosphorylate the Cdc27 component of the anaphase-promoting complex. Instead, these oocytes appear to enter S phase with subsequent DNA replication (87). Similarly, although PI3K activity is required for cell proliferation induced by a variety of mitogens, evidence suggests that the activation of PI3K is not sufficient for cell cycle progression (95). Indeed, PI3K may act in conjunction with MAPK signaling pathways to stimulate cell cycle progression and promote oocyte maturation (95). Thus, similar to the situation of MOS, although the activation of parallel or redundant MAPK-independent signaling pathways can substitute MAPK during oocyte meiotic resumption, it appears that MAPK still plays a major role in the G 2 /M transition under physiological conditions. One other aspect that merits mentioning is that, although full activation of MAPK is accompanied by MPF activation, Fisher et al. (86) find that a transient and low level of MAPK activation can be detected during progesterone treatment by using a highly sensitive method. This indicates that MAPK activation is an early event in response to progesterone, temporally dissociable from MPF activation. It is this low level of early activated MAPK that can initiate the powerful feedback loop that appears to be sufficient to induce meiotic maturation (this issue will be discussed in the topic of feedback loop). However, because this early MAPK activation is independent of protein synthesis, the pathways that lead to MAPK activation are still not clear. Apparently, additional studies are necessary to identify the potential physiological cascades responsible for meiotic resumption of oocytes in lower vertebrates. Mechanism of MPF Activation: Involvement of MAPK and Cyclin B Among various molecules, MPF plays the dominant role in the process of meiotic maturation. Immature Xenopus oocytes contain inactive pre-mpf that consists of cyclin B-bound Cdc2 phosphorylated on Thr14, Tyr15, and Thr161, as well as monomeric Cdc2 molecules that are about 10 times more than those bound to cyclin B (96, 97). In contrast to Xenopus, immature goldfish and Rana oocytes contain only monomeric Cdc2, and cyclin B is not detectable (98). As mentioned above, MAPK plays a dominant role in MPF activation in Xenopus oocytes, but this explanation can not reconcile the conclusion derived from goldfish and Rana because of the absence of pre-mpf in the oocytes of these species (88, 99). In fact, two mechanisms can account for MPF activation. The first one relies on the recruitment of monomeric Cdc2, which is combined with cyclin B but inactivated by the inhibitory kinase Myt1. Thus, the balance between the activities of Myt1 kinase and Cdc25C phosphatase play a pivotal role in the oocytes containing the pre-mpf. The second one relies on de novo synthesis of cyclin B, which may be sufficient to bind and activate Cdc2 that already is phosphorylated on T161 and therefore to generate a threshold level of Cdc2 activity that is able to trigger MPF autoamplification. For the first mechanism, Myt1 can be inhibited by either p90 rsk or MOS (89, 100). It has been shown that entopic expression of a constitutively active form of p90 rsk is sufficient to rescue the absence of MAPK activity in U0126-treated oocytes (87). Because Myt1 is the principal Cdc2-inactivating factor in oocytes, and p90 rsk is a physiologically relevant substrate for MAPK, a link exists between the MOS/MAPK cascade and MPF activation. Furthermore, Abrieu et al. (101) have found that MAPK activation slows the rate of Cdc2 inactivation in Xenopus and starfish oocytes, suggesting that MAPK either directly or indirectly brings about the inactivation of Myt1. Nevertheless, the MOS/MAPK pathway is able to promote MPF activation in the absence of cyclin B synthesis. Under this condition, the only possibility to form active MPF is to dephosphorylate Cdc2 of the pre-mpf store by Cdc25C. Other signal pathways independent of MAPK may also activate MPF by altering the balance of Myt1 and Cdc25C. For example, Akt can suppressively interact with Myt1 and mediate PI3K induced GVBD (94). It is also well known that polo-like kinase 1 (Plx1) is able to phosphorylate and activate the phosphatase Cdc25C, leading to dephosphorylation of Cdc2 and activation of MPF in Xenopus oocytes (102, 103). Collectively, these data indicate that pre-mpf can be activated through dephosphorylation of Cdc2 by inactivation of Myt1, activation of Cdc25C, or both. For the second mechanism, it is the newly synthesized cyclin B stimulated by steroids that is in charge of the activation of MPF. Thus, it is not difficult to understand why inhibition of MAPK could not block GVBD in goldfish and Rana oocytes (88, 99). Indeed, MOS/MAPK pathway may not serve to initiate meiotic maturation in animals in which pre-mpf is absent in immature oocytes. The de novo synthesis of cyclin B is necessary and sufficient to induce meiotic maturation in these species. However, accompanying the synthesis of cyclin B, another mechanism must exist to inhibit Myt1, which will suppress the newly formed MPF. Although artificial inhibition of MOS/MAPK does not affect GVBD, steroid-stimulated MOS synthesis and subsequent MAPK activation may participate in the inactivation of Myt1 under physiological conditions. In fact, it has been shown that coactivation of MPF and MAPK by injection of c-mos and cyclin B mrna promotes almost all of the morphological changes that occur during maturation without progesterone, whereas activation of one kinase without activation of

6 2042 Mol Endocrinol, September 2007, 21(9): Liang et al. Minireview the other induced only limited events (99). In addition, MAPK can phosphorylate cyclin B1 at one or more of the nuclear export signal phosphorylation sites (104). This phosphorylation is another way by which MAPK could positively influence Cdc2 activation and subsequent meiotic resumption. In addition to cyclin B, a novel protein termed Ringo (rapid inducer of G 2 /M in oocytes) or Speedy (rad1 mutant of Schizosaccharomyces pombe) was identified in an expression screen based on its ability to induce Xenopus oocyte maturation in the absence of progesterone stimulation (105, 106). Ringo/Speedy exercises its effects by binding and activating Cdc2, similar to the basic model of cyclin B. The relationship between Ringo/Speedy and other molecules such as MAPK is a novel field for future study. In summary, although results have shown that MPF has no species specificity, the quantity of MPF in the stockpile varies in different species. Thus, the mechanisms for activating MPF are somewhat different in various species. In some species, MAPK may induce MPF activation via preventing the activity of the inhibitory kinase, whereas in other species, the synthesis of cyclin B or Ringo/Speedy is necessary and sufficient for MPF activation. In most species such as Xenopus, these two pathways seem to be functionally redundant (107). MAPK-Related Feedback Loop Plays a Critical Role during Meiotic Resumption As a dynamic cellular unit, the intact oocyte has a more complicated signal transduction system than the linear sequence of enzymes described above. There is strong evidence that the positive feedback loop is important for regulating the all-or-none response of oocytes to the steroid stimulation during oocyte maturation. As important regulators of meiotic reinitiation, MAPK and MPF are involved in this powerful feedback loop. The function of MAPK is not restricted to affect its downstream target. More and more evidence demonstrates that activated MAPK can feedback to its upstream kinase MOS. For example, microinjection of activated forms of MEK and MAPK causes the accumulation of MOS, as long as protein synthesis is permitted (50, 108). The accumulation of MOS is attributable to both an increase in its synthesis and a decrease in its degradation (109). Accordingly, two mechanisms are used for the control of MOS by MAPK. First, MAPK stimulates mos mrna polyadenylation and thus stimulates MOS translation. When MAPK activity is inhibited, progesterone-induced mos mrna polyadenylation is attenuated (49). Second, MAPK inhibits MOS destruction, at least in part through phosphorylation of MOS at Ser3, a major site that can prevent MOS degradation (50, 110). In addition to MOS, complete phosphorylation of Raf requires the MAPK activity in both progesterone- and insulinstimulated meiotic resumption (75, 111). Thus, MAPKstimulated mos mrna cytoplasmic polyadenylation as well MOS and Raf phosphorylation are the key components of the positive feedback loop, which contributes to the all-or-none response of oocyte maturation. However, little is known about the mechanism regulating this powerful feedback loop. Recently, the scaffold molecule paxillin has been recognized as an essential regulator of the feedback loop in Xenopus leavis oocytes. Paxillin is required for accumulation of MOS protein and complete activation of downstream kinase signaling in response to steroids. Interestingly, paxillin activity also requires serine phosphorylation by MAPK (112). These data suggest that paxillin is an important regulator of the positive feedback effects of MEK/MAPK signaling on MOS protein accumulation. Similar to MAPK, MPF also participates in the feedback loop of MAPK activation and MOS accumulation. Microinjection of oocytes with active Cdc2 causes activation of MAPK by a protein synthesis-independent mechanism (113), implying that MAPK can be activated through a Cdc2-dependent mechanism. Moreover, injection of oocytes with a kinase-minus Cdc2 protein blocks MOS accumulation but does not affect the rate of MOS synthesis (114). The accumulation of MOS requires a stabilizing phosphorylation catalyzed by Cdc2 (17, 115). These findings suggest that MOS degradation is negatively regulated in a Cdc2-dependent manner. However, injection of cyclin B RNA or purified cyclin B protein induces both MOS protein accumulation and mos mrna polyadenylation, but this action requires MAPK activity. In contrast, the cytoplasmic polyadenylation of maternal cyclin B1 mrna was stimulated by MPF in an MAPKindependent manner, thus revealing a differential regulation of maternal mrna polyadenylation by the MAPK and MPF signaling pathways (49). The feedback loop is not limited to the interaction of MOS, MAPK, and MPF, because Cdc25C and Myt1 can also be regulated by MPF. A small amount of active MPF can bring about Cdc25C activation and Myt1 inactivation, thereby establishing a positive feedback loop (116). Furthermore, Plx1 can phosphorylate and activate Cdc25C in vitro (102); conversely, microinjection of Cdc25C brings about Plx1 activation, indicating that Cdc2, Plx1, and Cdc25C are all part of a positive feedback loop (117, 118). Summary of the Role of MAPK in Meiotic Resumption in Lower Vertebrates The need for MAPK during oocyte meiotic resumption in lower vertebrates has been debated for decades. Although it appears that many hormones, kinases, and phosphatases have the potential to induce MPF activation and subsequent GVBD, we still know very little about the physiological hormone and its receptor as well as biochemical events that play a physiological role during meiotic reinitiation. The new finding of testosterone as meiotic initiator indicates that androgens

7 Liang et al. Minireview Mol Endocrinol, September 2007, 21(9): may play a physiological role because they can induce meiotic resumption at a lower effective concentration. Therefore, our understanding of the beginning of oocyte meiotic resumption needs reconsideration. Because some preliminary results have shown that MAPK participates in androgen-induced GVBD, together with the role of MAPK in regulating the normal progression of steroid-triggered oocyte maturation, we may conclude that, under physiological conditions, MOS/MAPK pathway still plays the dominant role in oocyte maturation, although other signal pathways may substitute for the MOS/MAPK pathway to induce GVBD under certain artificial conditions. The evidence that p90 rsk phosphorylates and inactivates the Cdc2 inhibitory kinase Myt1 provides a link between MAPK and MPF. Other mechanisms also regulate MPF activation by changing the balance of Cdc25C and Myt1 or by producing the newly synthesized cyclin B. However, the relationship between MAPK and MPF is not a one-way action, because MPF can exert its effect on MAPK and other upstream proteins. Recent studies show that activation of a small amount of MAPK is an early event for steroid-induced meiotic resumption. If this is the case, combined with the powerful feedback loop, a possible mode is that MOS/MAPK can be activated through an MPF-independent pathway during the forepart of steroid stimulation. Then a small amount of MPF is activated by MOS/MAPK via changing the balance of Myt1 and Cdc25C. Once turning on active MPF, the MOS/MAPK pathway would positively regulate Cdc2 activation, creating a positive feedback loop. Then the vast activation of MAPK is accompanied with MPF activation during GVBD. This hypothesis can explain why MPF activation is delayed in the absence of MOS or MAPK activation and why MOS and MAPK injection is able to induce Cdc2 activation. Conversely, the positive feedback loop brings us a problem, i.e. it is not certain which component of the MAPK cascade is the initial target of the progesterone signal. Because a small change in the activity of components in this pathway will bring about the complete activation of the cascade, the initial molecular response to physiological steroid stimulation still needs additional investigation. The proposed functions and regulations of MAPK cascade in meiotic resumption of lower vertebrates are summarized in Fig. 1. THE ROLE OF MAPK ACTIVATION INSIDE MAMMALIAN OOCYTES DURING MEIOSIS RESUMPTION Intra-Oocyte MAPK Activation Cannot Be Detected before Meiotic Resumption in Mammals In mammals, the full amount of MAPK is synthesized in growing noncompetent oocytes. MAPK activation occurs during meiotic maturation in mammalian oocytes, and the kinetics of MAPK activation varies in different Fig. 1. Regulation of Oocyte Meiosis Resumption in Lower Vertebrates Progesterone-induced meiosis resumption in lower vertebrates is caused by alterations of several signal transduction pathways, including inactivation of camp-dependent PKA and activation of the MAPK pathway. 1) Under physiologycal condition, progesterone activates the signal transduction, which induces camp decrease, PKA inactivation, and subsequent protein synthesis of MOS. Then the cascade of MOS/MEK/MAPK activates MPF and initiates oocyte GVBD via p90 rsk activation and Myt1 inactivation. Besides, progesterone-induced MAPK activation can be accomplished through PI3K/Ras/Raf pathway. MAPK also plays a key role in androgen-induced meiotic resumption via PI3K or other unidentified pathways. Some growth factors, such as insulin, IGF-I, and FGF, can promote meiotic resumption through the pathway of Ras/Raf-induced MAPK activation. 2) In the absence of MAPK activation, other signal pathways that exert their roles on Cdc25C, Myt1, and cyclin B are important for activating MPF and inducing GVBD. The balance of Cdc25C and Myt1 can be conversed by MOS, Plx1, and PI3K/Akt pathways, whereas the de novo synthesis of cyclin B or Ringo/Speedy plays a key role in oocytes containing monomeric Cdc2. 3) In addition to the linear signal pathway, the powerful feedback loop plays a key role in oocyte maturation. MAPK can feedback to its upstream kinase MOS and Raf. MOS and MAPK activity is regulated by Cdc2 and cyclin B. Cdc25C and Myt1 can also be regulated by MPF.

8 2044 Mol Endocrinol, September 2007, 21(9): Liang et al. Minireview species. In mouse and rat oocytes, because the detected activation of MAPK occurs 2 h after spontaneous GVBD, it is proposed that MAPK is not required for spontaneous GVBD ( ). Indeed, in mouse denuded (DOs) and cumulus-enclosed (CEOs) oocytes, GVBD occurs normally when MAPK activation is prevented by the MEK inhibitor U0126 (124, 125). Our previous data also suggest that MAPK/p90 rsk activity is not required for GVBD in rabbit oocytes (126). Strong evidence supporting this hypothesis comes from studies using the mos-knockout (mos-ko) mouse model. In mos-ko oocytes, no activation of MAPK is detected while oocyte GVBD occurs normally, suggesting that activation of MAPK is not a prerequisite of GVBD (127, 128). Additional studies using the mos-deficient mouse model reveal that MOS/MAPK may play a crucial role in normal spindle and chromosome morphology as well as the reactivation of MPF after meiosis MI and thereafter the maintenance of MII arrest (129). Therefore, activation of MAPK within the oocyte is probably indispensable for postmeiotic resumption events rather than meiotic reinitiation in rodents. In oocytes from large domestic species, the temporal association between MAPK activation and GVBD is less defined. One reason is that the cytoplasm from most domestic animal oocytes is opaque because of the presence of dark lipid droplets, which makes it difficult to evaluate the nuclear status. Furthermore, meiotic resumption in domestic animal oocytes always lasts for a long duration, and thus the GVBD tends to occur asynchronously. These features make it more difficult to determine the correlation between meiotic resumption and MAPK activation in ungulate oocytes compared with rodent oocytes. In goat oocytes, the appearance of detected MAPK activity (10 12 h after maturation culture) is delayed compared with MPF activation and GVBD, which occurs 8 h after culture (130). In the bitch, MPF and MAPK activities are detected at low levels in oocytes at GV and GVBD stages and are significantly higher at MI and MII stages, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage) (131). In bovine oocytes, MAPK was activated at approximately the same time as GVBD (8 9 h of incubation) (132), but microinjection of MKP-1 mrna, which encodes a specific MAPK phosphatase, into GV-stage bovine oocytes can not prevent meiotic resumption (133). Increased MAPK activity in porcine oocytes is accompanied by the occurrence of GVBD. The previous study shows that microinjection of c-mos mrna into porcine oocytes promotes GVBD through the MPF pathway, but microinjection of porcine c-mos antisense RNA fails to arrest cells at the GV stage, although phosphorylation and activation of MAPK are completely inhibited throughout the maturation period (134). Our previous results indicate that antioxidants stimulate GVBD in the absence of MAPK activation (135). Besides, spontaneous meiotic resumption of porcine DOs occurs normally when MAPK phosphorylation is thoroughly inhibited by U0126 (136). Our recent study also shows that activation of MAPK in pig oocytes occurs after GVBD (137). All of these results suggest that, in domestic animals, detected MAPK activation is not implicated in the early events of meiosis resumption but rather in post-gvbd events. Artificial Activation of MAPK in Oocytes Stimulates GVBD Although MAPK activation can only be detected after mammalian oocyte meiotic resumption, under certain circumstances, artificial activation of MAPK can induce or promote GVBD. Fissore et al. (138) reported that, in bovine oocytes, injection of mos mrna elicited a rapid maximal activation of MAPK that resulted in accelerated resumption of meiosis, suggesting a positive effect of MAPK activation on GVBD. Another group found that, just before GVBD, part of the activated MAPK translocated into the GV and exogenous MAPK injected into the GV induced GVBD in porcine oocytes (139, 140). It has been reported that MAPK can be activated by MOS through two opposite pathways: activation of MEK1 or inhibition of phosphatases (141). Okadaic acid (OA), an inhibitor of phosphatase 1 and 2A, can induce MPF activation through inhibition of Cdc25 dephosphorylation in Xenopus oocytes ( ). Conversely, OA can also activate MAPK via an unidentified pathway and thus reverse the inhibitory effect of protein kinase C (PKC), camp, or 3-isobutyl-1-methylxanthine on GVBD in mammalian oocytes (121, 122, 145, 146). OA-activated MAPK induces the precocious activation of MPF as well as GVBD in both fully grown oocytes and growing oocytes (147, 148). It appears that an artificial increase in MAPK activity may be effective to induce the premature activation of MPF and GVBD. Besides, our previous study found that the ability of oocyte cytoplasm to activate MAPK is a prerequisite for GVBD competency (149). In porcine and rat oocytes that are incompetent to resume meiosis, as indicated by the failure of GVBD after extended culture, phosphorylation of MAPK and synthesis of cyclin B1 do not appear in vitro (121, 146). These results suggest that MAPK is an effective marker for evaluating the maturation competence of mammalian oocytes. All of the above data suggest that MAPK activation in oocytes may not directly regulate GVBD in mammals. In fact, it has been reported that Plk1 and Akt are involved in the MPF autoamplification loop, which is required and sufficient for meiosis resumption (150, 151). However, one aspect that merits mentioning is that mammalian oocytes are much smaller than Xenopus oocytes. Undoubtedly, mammalian oocytes may contain significantly less MAPK than Xenopus oocytes, and the amount of MAPK that is actually phosphorylated during mammalian oocyte maturation could be significantly less. It is therefore plausible to postulate that, much like in Xenopus, a small amount of MAPK that contributes to the feedback loop might

9 Liang et al. Minireview Mol Endocrinol, September 2007, 21(9): be activated before GVBD under physiological conditions but simply cannot be detected by conventional Western blot analysis or kinase assays. Additional studies are therefore necessary to test this possibility using a much more sensitive, yet waiting to be developed, method for detection of MAPK activation. Nevertheless, despite the fact that meiosis resumption of mammalian oocytes can be induced through MAPKindependent pathways, MAPK is involved in spindle formation, MI to MII transition, and MII arrest (29, 30). THE ROLE OF MAPK ACTIVATION IN FOLLICULAR SOMATIC CELLS IN MEIOSIS RESUMPTION Recent studies provided new insights into the role of MAPK-dependent pathways in meiotic resumption of mammalian oocytes: MAPK activity in follicular somatic cells is necessary for gonadotropin-induced meiotic resumption in oocytes. In the following section, we will discuss the potential role of MAPK signal pathway in follicular somatic cells, especially cumulus cells, in the induction of mammalian oocyte meiotic resumption. MAPK Activation in Follicular Somatic Cells Is Necessary for Gonadotropin-Induced Mammalian Oocyte GVBD It is well known that the preovulatory LH surge induces the resumption of meiosis in mammalian oocytes by propagating its action to follicular somatic cells, including granulosa cells and cumulus cells (152). However, the signal pathways occurring thereafter in follicular somatic cells responsible for the induction of oocyte GVBD is not clear. Several recent studies reveal that activation of MAPK in cumulus cells is probably required for gonadotropin-induced meiotic resumption in mammalian oocytes. When rodent and porcine CEOs were cultured in vitro, FSH induced MAPK (including p38 MAPK) activation before GVBD in cumulus cells (124, 137, ), and the selective MEK inhibitors PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or U0126 block FSH-induced meiotic resumption in CEOs but not spontaneous meiotic resumption in DOs (124, 136, 137, 156). In mos-ko mice, no MAPK activity is detected in oocytes, but, in cumulus cells, activation of MAPK is detected before gonadotropin-induced GVBD both in vivo and in vitro. Moreover, inhibition of MAPK activation blocks FSH-induced oocyte GVBD in cultured CEOs (153). Because MAPK is not activated in oocytes, the meiosis inhibitory effect of MAPK inhibitor must target MAPK in cumulus cells. This observation supports the hypothesis that MAPK activation in cumulus cells is essential for the induction of oocyte meiotic resumption. Inhibition of MAPK activation also blocks several other key processes normally occurring in cumulus cells after ovulatory stimulation, such as cumulus expansion and Has2 (hyaluronan synthase 2), Ptgs2 (prostaglandin-endoperoxide synthase 2) expression (157). Thus, the gonadotropin-induced oocyte meiotic resumption is probably mediated by the MAPK-dependent pathway in cumulus cells. In addition to cumulus cells, activated MAPK in granulosa cells also plays a major role in mediating LH function. In porcine granulosa cells, MAPK is activated immediately after the administration of LH and FSH (158). Moreover, the model of whole follicle culture that more closely resembles physiological conditions has been used to test the role of MAPK. In both mouse and rat, inhibition of MAPK activity prevents LH-stimulated meiosis resumption in follicle-enclosed oocytes. In cultured rat granulosa cells, inhibition of MAPK significantly prevents human chorionic gonadotropin-induced Runx1 (runt-related transcription factor 1) mrna expression (159). These results indicate that the MAPK-dependent pathway in granulosa cells plays key roles during the induction of oocyte meiotic resumption (160). Because MAPK activation occurs immediately in granulosa cells rather than in the oocyte after exposure of rat follicles to LH, the meiotic inhibitory effect of MAPK inhibitor is probably brought about by blocking MAPK activation in granulosa cells (160). Moreover, when granulosa cells or CEOs were treated with U0126, gonadotropin-induced progesterone production was significantly downregulated, whereas the level of estradiol was significantly upregulated (161). These results suggest that the LH-induced differential synthesis of progesterone and estradiol in mural and cumulus granulosa cells is mediated by an MAPK-dependent pathway. Induction of Oocyte Meiotic Resumption by Activation of MAPK in Follicular Somatic Cells: Potential Mechanisms The mechanism for inducing oocyte meiotic resumption after MAPK activation in cumulus cells is not clear. If MAPK functions by inducing gene transcription and then protein synthesis in cumulus cells, treatments that block gene expression at the translation level are expected to abolish the effect of MAPK activation. This was exactly the case in our previous experiments in which we found that protein synthesis inhibitor cycloheximide prevented FSH- or phorbol 12-myristate 13-acetate-induced oocyte meiotic resumption (162). Thus, one potential mechanism of the follicular somatic cell-mediated induction of oocyte GVBD would be the MAPK-dependent production of a putative positive meiosis resumption-inducing (activating) factor (152, 163). Meiosis activating sterol (MAS) might be a suitable candidate, because follicular purine-inhibited meiosis resumption of CEOs can be overcome by a diffusible meiosis-inducing substance secreted by the cumulus cells (164), which has been identified as MAS (165). Furthermore, MAS can stimulate GVBD in mouse and porcine oocytes ( ), but other re-

10 2046 Mol Endocrinol, September 2007, 21(9): Liang et al. Minireview ports doubt the function of MAS in stimulating GVBD because of its restricted role in physiological meiotic resumption ( ). More recent studies show that steroid is another candidate. In mouse, steroids are secreted from granulosa/cumulus cells under the stimulation of EGF, and steroids can combine with their receptors and thus trigger the occurrence of GVBD at lower concentrations compared with gonadotropin (174, 175). It suggests that steroids might play a physiological role in normal meiotic resumption. However, still more direct evidence is needed to prove this hypothesis, and additional effort is required to identify the putative MAPK-stimulated meiosis resumption-inducing factors during oocyte maturation. Some reports suggest that disruption of gap junctional communication between the oocyte and cumulus/granulosa cells might be part of the mechanism that induces oocyte meiotic resumption after MAPK activation in follicular somatic cells. Gap junctions present a major communication system between the oocyte and its associated cumulus/granulosa cells in intact ovarian follicles. These gap junctions play important roles in promoting oocyte and follicle development, maintaining oocyte meiotic arrest, and inducing oocyte meiotic resumption (152, 176). The maintenance of oocyte meiotic arrest within Graafian follicles requires a signal sent to the oocyte via gap junctions from follicular somatic cells (152). Hence, the reduction or the disruption of functional gap junctions between follicular somatic cells and the oocyte would induce oocyte meiotic resumption (177). Gap junction protein -1, also commonly known as connexin (Cx) 43, is the major component of gap junctions between ovarian granulosa cells (178, 179). Cx43 can be phosphorylated by several kinases, such as PKA, PKC, glycogen synthase kinase 3, and MAPK, and its phosphorylation results in disruption of gap junctions (160, 180). In rat whole follicle culture, LH induces the immediate phosphorylation of Cx43, followed by its down-regulation in granulosa cells, which precedes GVBD (181). In addition, blocking gap junctional communication within the follicle leads to oocyte GVBD (182). These observations suggest that disruption of gap junctions within the follicle mediates LHinduced oocyte meiotic resumption in the rat. The inhibition of MAPK activation blocks oocyte GVBD and early phosphorylation of Cx43, suggesting that the MAPK-dependent pathway mediates LH-induced breakdown of gap junctional communication and thus leads to oocyte meiotic resumption (181). Because activation of MAPK in rat granulosa cells is detected earlier than in the oocyte and is detected before GVBD (160), the phosphorylation of Cx43 and subsequent disruption of gap junctional communication between follicular somatic cells and the oocyte is probably mediated by activation of MAPK. Although Cx43 has been shown to be localized in rat oocytes (183), the main gap junctional component that functions between cumulus cells and oocytes is Cx37 ( ). Until now, few experiments have been conducted to clarify the relationship between MAPK and Cx37. Studies of this topic will be helpful in clarifying the interaction between follicle cells and oocytes. PKC and camp-dependent PKA Participate in the Activation of MAPK in Cumulus Cells The upstream pathway of MAPK in follicular somatic cells is less known. Because some of the biochemical steps linking GPRs to MAPK activation include PKC (188) and camp-dependent PKA (189, 190), the pathways related to these two kinases probably mediate the activation of MAPK in follicular somatic cells. PKC plays roles in both oocytes and follicular somatic cells. We reported that activation of PKC significantly down-regulated MAPK phosphorylation and inhibited meiosis resumption in DOs (121, 149, 191), but, in CEOs, the effects on meiotic resumption appear to be positive because PKC activators phosphorylate MAPK in cumulus cells and induce GVBD of CEOs in the absence of FSH stimulation (162, 192). Similarly, in HX-supplemented medium, PKC and intracellular calcium (which is a well-known activator of conventional PKC isoforms) are involved in FSH-mediated GVBD of porcine CEOs (193). The downstream events of PKC in cumulus cells that lead to oocyte meiotic resumption have been investigated in our previous studies. We showed that PKC inhibitors block FSH-induced oocyte meiotic resumption and MAPK activation, whereas FSH and PKC activator-induced GVBD can be reversed by U0126. It appears that PKC might be the link between FSH stimulation and MAPK activation in meiotic resumption of mouse CEOs (162). In granulosa cells, sustained activation of MAPK is dependent on PKC activity, whereas inhibition of PKC activity is associated with attenuated phosphorylation of MAPK (194). Because one of the ultimate effects of MAPK activation in follicular somatic cells is inducing the breakdown of gap junctions, PKC pathway may also exert its effect on the interruption of gap junctions in follicular somatic cells (195). There is abundant evidence showing that the second-messenger camp and camp-dependent PKA play an important role in controlling meiotic resumption of oocytes (8, ). Similar to PKC, the role of camp-pka in the control of meiosis appears different in oocytes and follicular somatic cells. In oocytes, it was reported that PKA can activate Wee1B (Wee1-like protein kinase 1B) or inhibit Cdc25B, which downregulates MPF activity and prevents GVBD (200, 201) (Yu, B. Z., personal communication), whereas in granulosa/ cumulus cells, camp-dependent PKA has a positive effect on MAPK activation as well as oocyte meiotic resumption (137, 157, 158). More recent studies have shown that LH-, human chorionic gonadotropin-, or FSH-stimulated MAPK activation is mediated by camp-dependent PKA in follicular somatic cells, and LH-inhibited Cx43 expression is mediated by both PKA and MAPK (153, 181, ). It appears that, in follicular somatic cells, activation of MAPK is one of

11 Liang et al. Minireview Mol Endocrinol, September 2007, 21(9): the downstream events of camp-dependent PKA activation. To clarify this hypothesis, the crosstalk between camp and MAPK in cumulus cells was investigated by using cell-type-specific PDE isoenzyme inhibitors in our recent study. The results show 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 (137). Link between LH Signaling and MAPK Activation in Follicular Somatic Cells: Participation of EGF- Like Growth Factors LH-induced oocyte meiotic resumption is dependent on the activation of MAPK in follicular cells. However, the mediator between LH stimulation and MAPK activation has been an enigma for a long time. It has been proposed recently that the EGF network may play an important role in mediating LH function during oocyte meiotic resumption. For example, EGF and EGF family members amphiregulin (AREG), epiregulin (EREG), and -cellulin influence meiotic maturation and developmental competence of oocytes in various mammalian species, and incubation of follicles with these growth factors recapitulates the morphological and biochemical events triggered by LH (12, 13, 205). Similarly, inhibition of the EGF receptor (EGFR) kinase or depletion of AREG or EREG prevents EGF-induced steroidogenesis and blocks LH-induced steroidogenesis as well as meiotic resumption (175, 206). This implicates that EGF family members may have a physiological role in the regulation of meiotic reinitiation in preovulatory follicles, presumably as a mediator of signals elicited by the LH surge and thus is critical for normal gonadotropin-induced meiotic resumption in female gonads. The expression of EGFR in zebrafish is mainly restricted to the follicular somatic cells with little expression in the oocytes (207). In goat, although EGFR is detected in oocytes, its phosphorylation during the maturation process can not be observed (208). In the mouse, EGF family members overcome HX-inhibited meiotic resumption only in CEOs but not DOs (13). These results indicate that follicular somatic cells but not oocytes are the major sites for EGFR to play its role. Recently, the model of triple-membrane-passing signaling has emerged, which can well explain the role of EGF family members in gonadotropin-mediated meiotic resumption in mammalian oocytes (209, 210). In this model, LH binds to the LH receptor on granulosa cells of preovulatory follicles and thus activates the camp- and p38 MAPK-dependent signal cascades and stimulates the expression of mrnas encoding AREG, EREG, and -cellulin (211). These preform EGF family members are processed into mature peptides by metalloproteinase and released from the cell surface. Subsequently, the soluble growth factors exert autocrine and paracrine effects through activation of EGFR and other signaling in granulosa cells and cumulus cells. All of these processes will lead to oocyte meiotic resumption, cumulus expansion, and events that are crucial for ovulation. After EGF binding, the activated EGFR, which possesses an intrinsic tyrosine kinase, participates in the phosphorylation of numerous tyrosine kinase substrates within cells (212). One recently discovered pathway of EGF signal transduction in the ovary involves the rapid phosphorylation of MAPK. For example, in porcine and rabbit granulosa cells, MAPK can be activated after a transient treatment with EGF (158, 213, 214). In cumulus cells, EGF acts on bovine CEOs from small follicles to accelerate the meiotic cell cycle of oocytes. This accelerating effect may be related to MAPK activity during the early stages of maturation (215). Moreover, exposure of mouse GV-stage CEOs to EGF induces a considerable increase in MAPK phosphorylation (216, 217). Conversely, AG 1478 [4-(3-chloro-anilino)-6,7-dimethoxyquinazoline], an inhibitor of the EGFR, suppresses EGF-stimulated phosphorylation of MAPK. Treatment with the MEK inhibitor PD98059 or U0126 abolishes EGF-induced MAPK activation as well as GVBD (208) (our unpublished data). All of these results indicate that activation of EGFR triggers signaling via the MAPK pathway and that EGF mainly acts on follicular somatic cells to regulate oocyte maturation. The functional role of EGF during oocyte maturation has been partially revealed in recent studies. EGF family members can promote cumulus cell expansion via their stimulative effect on Has2, Ptgs2 mrna expression, and this effect appears to be mediated by MAPK (13, 217). Furthermore, EGF has the ability to induce the steroidogenesis in follicular somatic cells (175). Coincidentally, the process of LH-induced steroidogenesis is also dependent on MAPK activity in both cumulus and granulosa cells. Thus, the interaction between EGF and MAPK during steroidogenesis is another topic that merits investigation. Finally, how does EGF activate MAPK? If other molecule(s) mediate EGF-induced MAPK activation, identification of these candidates will be helpful for exploring the whole story of this important reproductive event. Summary of the Role of MAPK in Meiotic Resumption in Mammalian Oocytes Although it appears that MAPK activation within oocytes is not essential for MPF activation and GVBD, artificial activation of MAPK could reinitiate meiosis resumption. It is possible that the initial small amount of active MAPK, although not detected by current conventional method, might play a role during GVBD, even if it can not be detected by currently available conventional methods. In any case, quite a number of recent studies provide evidence that MAPK activation in follicular somatic cells is indispensable for oocyte meiotic resumption. The role of MAPK may be achieved or regulated by meiosis resumption-inducing

12 2048 Mol Endocrinol, September 2007, 21(9): Liang et al. Minireview Fig. 2. Regulation of Oocyte Meiosis Resumption in Mammals In mammalian oocytes, Plk1- and Akt-induced MPF activation plays a key role in the events of GVBD. MPF activity can be inhibited by PKC or by camp-dependent PKA via Cdc25B and Wee1B pathway. This mechanism is independent of MAPK activation inside oocytes, but artificial activation of MAPK inside oocytes is able to induce MPF activation and thus leads to GVBD. Conversely, MAPK in follicular somatic cells is necessary for gonadotropin-induced meiotic resumption. Gonadotropin-induced activation of GPR in granulosa cells leads to activation of p38 MAPK and camp-dependent PKA, which promotes the expression of pre-egf family members. After splitting of these preforms of growth factor by metalloproteinase (MMP), the matured EGF family members induce the activation of EGFR in granulosa/cumulus cells and thus activate MAPK through an unidentified pathway. FSH stimulates the FSH receptor located on the cumulus cell membrane and then activates MAPK via PKC and camp-dependent PKA pathway. One possible role for activated MAPK in granulosa/cumulus cells is to promote the expression of MAS, which translocates from cumulus cells to oocytes via gap junctions and induces MPF activation. The other possible role is to stimulate the synthesis of steroids that combine with the receptor located on oocyte and induce oocyte MAPK activation. Activated MAPK in granulosa/cumulus cells can also induce inactivation of Cx37 and Cx43 in follicular somatic cells and oocytes, respectively, and thus terminate the transfer of camp from the somatic cells to the oocyte. All of these events induce the reinitiation of meiotic maturation in mammalian oocytes.