cAMP-Dependent PKA Negatively Regulates Polyadenylation of cmos mRNA in Rat Oocytes

Activation of members of the MAPK family, Erk 1 and 2, in oocytes resuming meiosis is regulated by Mos. The cAMP-dependent PKA-mediated cAMP action that inhibits the resumption of meiosis also prevents MAPK activation. We hypothesized that PKA interferes with the MAPK signaling pathways at the level of Mos. We also assumed that this regulatory cascade may involve p34cdc2. To test our hypothesis we explored the role of PKA and p34cdc2 in regulating Mos expression. Rat oocytes that resume meiosis spontaneously served as our experimental model. We found that meiotically arrested rat oocytes express the c-mos mRNA with no detectable Mos protein. The presence of Mos was initially demonstrated at 6 h after meiosis reinitiation and was associated with its mRNA polyadenylation. (Bu)2cAMP inhibited Mos expression as well as c-mos mRNA polyadenylation. Both these cAMP actions were reversed by the highly selective inhibitor of the catalytic subunit of PKA, 4-cyano-3-methylisoquinoline. Polyadenylation of c-mos mRNA was also prevented by roscovitine, which is a potent inhibitor of p34cdc2. Ablation of MAPK activity by two specific MAPK signaling pathway inhibitors, either PD 98059 or U0126, did not interfere with Mos accumulation. Our results suggest that translation of Mos in rat oocytes is negatively regulated by a PKA-mediated cAMP action that inhibits c-mos mRNA polyadenylation and involves suppressed activity of p34cdc2. We also demonstrate that stimulation of Mos synthesis in the rat does not require an active MAPK. (Molecular Endocrinology 16: 331–341, 2002)

M EIOSIS IN THE oocyte is initiated during fetal life and is arrested at prophase.Oocytes that exit from prophase arrest undergo germinal vesicle breakdown (GVB) and chromosome condensation, proceed to the first metaphase (MI), emit the first polar body (PBI), and arrest again at the second metaphase (MII).The series of events that starts with GVB and ends at MII leads to the production of a mature fertilizable oocyte and is therefore defined as oocyte maturation.This process is subjected to regulation by the maturation promoting factor (MPF) composed of p34cdc2, a 34-kDa Ser/Thr kinase, and a 45-kDa B1 type cyclin (1).Members of the MAPK family, Erk 1 and 2, also participate in regulation of meiosis (2,3).The signal for MAPK activation is mediated by a MAPK kinase (MEK), which in oocytes is activated by Mos (4).
The exclusive expression of c-mos protooncogene in germ cells (5)(6)(7)(8) suggests a specific function for its 4lkD protein product in the meiotic cell cycle.Indeed, in Xenopus, Mos has been identified as a key regulator of meiosis that acts at multiple stages along this process.In oocytes of this animal species Mos is required for stimulation of entry into the first meiotic division (9,10) and for suppression of DNA replication between the first and the second rounds of meiosis (11).Mos in amphibians is also an active component of the cytostatic factor that holds unfertilized eggs arrested at MII (12).On the other hand, targeted disruption of c-mos gene in embryonic stem cells to generate mos-deficient mice (13,14) seems to indicate that Mos kinase in mammals is necessary only for MII arrest.Analysis of oocytes of these mutant mice revealed that their capacity to reinitiate meiotic maturation and their potential to progress through the first to the second metaphase were not affected.Furthermore, DNA replication between the two rounds of meiosis in these mice remained suppressed.However, their ability to arrest at MII was severely impaired, resulting in spontaneous parthenogenetic activation (15).Other reports suggest that Mos kinase in mouse oocytes may also participate in regulation of spindle formation (16)(17)(18)(19).
Despite these and other differences between amphibian and mammalian oocyte maturation, it is well accepted that meiosis in oocytes of both animal species is subjected to negative regulation by the cAMPdependent PKA (20,21).The phosphoprotein that serves as a substrate for PKA in vertebrate oocytes has not been identified as yet.However, demonstrations that experimental manipulation of either intraoocyte concentrations of cAMP or PKA activity within the oocyte interferes with MAPK activation (22)(23)(24) seem to suggest that the MAPK signaling cascade may be subjected to regulation by the cAMP/PKA pathway.
Another report of PKA inhibition of Mos-induced oocyte maturation (25) is consistent with this idea.Taking these findings into consideration, we assumed that the cAMP signal transduction pathway may obstruct the MAPK signaling cascade in oocytes resuming meiosis at the level of Mos.Our study was designed to test this hypothesis.Our experiments revealed that a PKAmediated cAMP action negatively regulates the translation of Mos, further demonstrating for the first time that this effect is elicited by inhibition of mos mRNA polyadenylation.We also show that in rat oocytes activation of p34cdc2, but not MAPK, is a prerequisite for Mos expression.

Expression of Mos in Rat Oocytes Throughout Meiotic Maturation
Prophase-arrested oocytes recovered from the ovarian follicles are characterized by the presence of germinal vesicles (GV).Spontaneous reinitiation of meiosis in vitro that is morphologically detected by GVB takes place in our rat colony at 2-6 h of culture (Fig. 1A).This event is accompanied by chromosome condensation and spindle formation (data not shown).The first meiotic division is completed by the emission of PBI that occurs at 12 h of incubation (Fig. 1A) and is immediately followed by MII.The oocytes remain arrested at MII for at least 24 h of incubation.
Our first experiment was directed at the analysis of c-mos gene expression in fully grown rat oocytes throughout meiosis.Meiotically incompetent, growing oocytes, were also examined.Using RT-PCR we found that prophase-arrested both meiotically incompetent and competent oocytes contain c-mos mRNA.No modulations of this transcript were observed in fully grown oocytes throughout meiosis.Identification of the RT-PCR product was confirmed by sequence analysis that revealed 100% identity to the c-mos gene.Elevated intraoocyte cAMP concentrations obtained by the addition of (Bu) 2 cAMP (2 mM) together with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (20 M), to the culture medium had no effect on c-mos gene expression (Fig. 1B).
Despite the presence of the c-mos transcript Western blot analysis failed to demonstrate its protein product in prophase-arrested oocytes for the first 4 h of culture.Mos could be initially detected at 6 h of incubation, which is after GVB.The amount of Mos was substantially elevated at MI of meiosis (8 h of incubation) and remained high in the MII-arrested oocytes after an overnight incubation (Fig. 1C).
The kinetics of Mos accumulation temporally corresponds to the pattern of MAPK activation.As shown in Fig. 1D, basal activity of MAPK was observed during the initial 6 h of incubation with an increase of enzymatic activity at 8 h.MAPK activity reached its maximum at 12 h of incubation and remained high in MIIarrested oocytes.Another group of MII oocytes that were hormonally stimulated to resume meiosis in vivo was recovered from the oviductal ampulla after ovulation.These oocytes also exhibited a level of MAPK activity that was comparable to that of MII oocytes that underwent meiotic maturation spontaneously in vitro.
The abundance of Mos at any given time point apparently represents the ratio between the synthesis and degradation of this protein.Mos degradation is brought about by the ubiquitin-proteasome pathway (26).Nevertheless, the undetectable amounts of Mos at the early stages of meiosis do not reflect its extensive proteolysis since incubation of oocytes with MG132 (10 M), a potent inhibitor of proteasomal catalytic activity, did not affect the content of Mos in the oocytes (Fig. 2A).Degradation of cyclin B1, which is another substrate for the ubiquitin-proteasome pathway, has been shown by us recently to be effectively inhibited by this concentration of MG132 (27).The demonstrated resistance of Mos to degradation at early stages of meiosis, combined with the failure of oocytes incubated with the protein synthesis inhibitor, cycloheximide (100 g/ml), to express Mos (Fig. 2B), provide confirming evidence to the idea that the expression of this kinase at early stages of meiosis is regulated at the level of translation.

Effect of cAMP-Mediated PKA Action on Mos Expression
To test the possible effect of cAMP on Mos expression, oocytes were incubated under conditions that elevated intracellular concentrations of this cyclic nucleotide.We found that accumulation of Mos was prevented by the combined effect of (Bu) 2 cAMP (2 mM) and the cAMP phosphodiesterase inhibitor, IBMX (20 M).Removal of these agents resulted in a full recovery of their inhibition within an additional incubation time of 6 h (Fig. 3A).Experimental elevation of intraoo-

Fig. 2. Mos Accumulation Is Inhibited by Cyclohexamide
A, The effect of MG132 on Mos accumulation.Oocytes were incubated in the presence or absence of MG132 (10 M) for 4 and 8 h.Western blot analysis for the presence of Mos was performed as described for Fig. 1C.The results of one representative experiment of three repetitions are presented.B, The effect of the protein synthesis inhibitor, cycloheximide (CHX), on Mos expression.Oocytes (500/lane) were incubated in the presence or absence of CHX (100 g/ml) for 8 h.Western blot analysis for the presence of Mos was performed as described for Fig. 1C.The results of one representative experiment of three repetitions are presented.cyte concentration of cAMP also resulted in inhibition of MAPK activation (Fig. 3B).
In a previous study we have shown that cAMP concentrations in rat ooyctes reach minimal levels at 45 min after their isolation from the ovarian follicles (28).On the other hand, we show herein that Mos accumulation that is inhibited by cAMP cannot be detected before 6 h of incubation.The extended time interval that elapses between the drop in intraoocyte cAMP and mos translation seems to raise some concerns in regard to the direct correlation between these two events.To resolve this apparent conflict, we analyzed the cAMP-sensitive initial step for Mos expression using the following experimental strategy.Freshly isolated oocytes were transiently exposed to (Bu) 2 cAMP/ IBMX-free medium for varying periods of time.The oocytes were then transferred into medium containing these cAMP elevating agents for further incubation.After a total incubation time of 8 h, the oocytes were analyzed for the presence of Mos.As shown in Fig. 4, a 45-but not 30-min incubation in cAMP-free medium allowed the oocytes to express Mos.These results suggest that initiation of Mos expression in rat oocytes take place between 30 and 45 min after their isolation from the ovarian follicle.They further provide strong evidence for the direct correlation between the oocyte maturation-associated drop in intraoocyte cAMP and mos translation.
To test whether the inhibitory effect of cAMP on Mos expression is mediated by PKA, we further tested the influence of 4-cyano-3-methylisoquinoline (4C3M), which is a highly selective and most potent inhibitor of the catalytic subunit of PKA (29).The sensitivity range of PKA activity to this agent was characterized in rat granulosa cell lysate.Addition of 4C3M to a PKA phosphorylation assay resulted in a dose-dependent inhibition of PKA activity (Fig. 5A).We found that the maximal effective dose of 4C3M (200 nM) also reversed the negative effect of cAMP on Mos accumulation (Fig. 5B).
The translation of mos in oocytes is mediated by its polyadenylation (30).To explore the mode of regula-

Fig. 3. Inhibition of Mos Accumulation and MAPK Activation by cAMP
A, The effect of cAMP on Mos accumulation.Oocytes (500/lane) were incubated for 6 h in the presence and absence of (Bu) 2 cAMP (2 mM) and IBMX (20 M) in the culture medium.When indicated, removal of (Bu) 2 cAMP and IBMX was followed by additional incubation for 6 h in inhibitor-free medium.Western blot analysis was performed as described for Fig. 1C.The results of one representative experiment of three repetitions are presented.B, The effect of cAMP on MAPK activation.Oocytes were incubated in the presence or absence of IBMX (0.2 mM) in the culture medium.The oocytes (50/lane) were extracted, electrophoretically separated, and immunoblotted with anti-P-MAPK and anti-total MAPK antibodies as described for Fig. 1C.The results of one representative experiment of five repetitions are presented.

Fig. 4. Timing of cAMP-Sensitive Initial Step for Mos Expression
Freshly isolated oocytes (500/lane) were exposed to (Bu) 2 cAMP/IBMX-free medium for varying periods of time.The oocytes were then transferred into medium containing (Bu) 2 cAMP (2 mM) and IBMX (20 M) for further incubation.After a total incubation time of 8 h, Western blot analysis for the presence of Mos was performed as described for Fig. 1C.The results of one representative experiment of three repetitions are presented.tion of c-mos mRNA polyadenylation and to specifically examine the possible interference of cAMP with this process, the poly(A) test assay was employed (31).Using this assay we found that in prophase-arrested oocytes the c-mos mRNA possesses a poly(A) tail of about 50 bp.No changes in its length could be demonstrated in oocytes incubated for 4 h.An increase of about an additional 100 bp in the length of the poly(A) tail of c-mos mRNA was observed at 6 h with a further elongation at 8 h of incubation.Polyadenylation of c-mos mRNA could not be demonstrated in oocytes incubated with (Bu) 2 cAMP and IBMX.However, the inhibitory effect of (Bu) 2 cAMP on c-mos mRNA polyadenylation was effectively reversed by the PKA inhibitor, 4C3M (200 nM, Fig. 6A).
Cytoplasmic polyadenylation in Xenopus oocytes stimulates cap ribose methylation (32).A more recent study further demonstrated that cap ribose methyl-  ation of c-mos mRNA in these oocytes stimulates translation (33).The involvement of cap ribose methylation in rat oocytes was tested in our system using the methyl transferase inhibitor, S-isobutyl-thio-adenosine [SIBA (31)].As shown in Fig. 6B, inhibition of c-mos mRNA cap ribose methylation prevented Mos accumulation.

p34cdc2 Participates in Regulation of Mos Translation
A previous study in our laboratory demonstrated that dephosphorylation and thus activation of p34cdc2 is negatively regulated by cAMP (34).We show herein that elevated intraoocyte concentrations of cAMP effectively prevent c-mos mRNA polyadenylation.Taken together, these observations may suggest that an active p34cdc2 is involved in recruitment of c-mos mRNA for translation and elongation of its poly A tail.This assumption was examined in rat oocytes incubated with roscovitine, a purine analog that selectively inhibits p34cdc2 (35).We have recently demonstrated that, at a concentration of 100 M, this agent prevented MPF activation and abolished Mos expression (Ben-Yhoshua Josefsberg, L., D. Galiani, S. Lazar, O. Kaufman, R. Seger, and N. Dekel, unpublished).As shown in Fig. 7, a similar concentration of roscovitine totally blocked c-mos mRNA polyadenylation.

Mos Expression Is Independent of an Active MAPK
Translation of Mos that requires an active MAPK has been suggested for Xenopus oocytes (23).To test this possibility in a mammalian system, we incubated rat oocytes in the presence of PD 98059, a highly selective MEK inhibitor (37).As shown in Fig. 8A, a concentration of PD 98059 that effectively inhibited MAPK activity failed to affect the accumulation of Mos in rat oocytes (Fig. 8B).These findings were further confirmed by the use of U0126, another potent and specific MEK inhibitor (38).Both these compounds share a common or overlapping binding site on MEK that is clearly distinct from those of ATP and MAPK.However, U0126 affinity for MEK is approximately 100-fold higher than that of PD 98059 (38).As expected, oocytes incubated in the presence of U0126 did not exhibit an active MAPK.Confirming the results of our previous experiment, these conditions did not interfere with accumulation of Mos (Fig. 8, C and D).Taken together, our findings deny a role for MAPK in stimulating Mos synthesis, disclosing an additional dissimilarity between the mode of regulation of meiosis in mammalian and amphibian oocytes.

DISCUSSION
It has been more then 20 yr since the mediatory role of PKA in eliciting the negative effect of cAMP on reinitiation of meiosis was initially reported in Xenopus oocytes (20).In addition, the fact that an active PKA is responsible for maintenance of oocytes in prophase arrest has been confirmed in mice almost 10 yr later (21).However, the phosphoprotein that serves as a substrate for PKA in vertebrate oocytes remained unknown, and the downstream events subjected to regulation by the cAMP/PKA signal transduction pathway are still not identified.Demonstrations that microinjection of the catalytic subunit of PKA into Xenopus oocytes prevents MAPK activation (39) raised the intriguing idea that either this enzyme and/or any of its upstream regulators, such as Mos, could possibly serve as target(s) for PKA action.
The presently available information regarding the interference of PKA with Mos signaling cascade is controversial.An earlier study presents evidence that Mos translation in Xenopus is blocked by PKA (39), whereas a later report claims that inactivation of PKA is not required for c-mos translation during meiotic maturation of amphibian oocytes (23).The only study addressing this issue in a mammalian system demonstrates that in mouse oocytes it is not Mos synthesis, but rather its activity, that remains inhibited by PKA (40).
Overcoming this apparent confusion, our study clearly demonstrates that elevated intraoocyte concentrations of cAMP effectively prevented mos translation and that this effect was accompanied by inhibition of c-mos mRNA polyadenylation.It further shows that the suppression of PKA catalytic activity fully reversed these inhibitory effects of cAMP.Taken together, our results provide the first evidence that translation of mos in rat oocytes is negatively regu- lated by a PKA-mediated cAMP action that inhibits c-mos mRNA polyadenylation.Recent observations that have indicated that PKA does not necessarily account for all the intracellular targets of cAMP (41) accentuate the significance of these findings.
Suppression of Mos accumulation by experimental elevation of intraoocyte cAMP concentration has also been demonstrated in progesterone-treated Xenopus oocytes (23).However, IBMX in this study failed to inhibit Mos synthesis in oocytes microinjected with recombinant c-mos.These investigators concluded therefore that MAPK activation, rather than PKA inactivation, is required for the onset of c-mos translation.A positive feedback loop between MAPK and Mos during Xenopus oocyte maturation has been also proposed previously (2,42,43).In accordance with this idea, a more recent study further suggested a role for MAPK in stimulating c-mos mRNA cytoplasmic polyadenylation during Xenopus oocytes maturation (44).
In disagreement with the above mentioned observations, our findings show a substantial accumulation of Mos under conditions of inhibited activity of MAPK.
These results suggest that in the rat, MAPK activation is not a prerequisite for the c-mos mRNA polyadenylation and for the onset of Mos translation.Analysis of the mos-deficient murine oocytes (15) presented the different repertoire of events throughout meiosis regulated by the Mos/MAPK signaling pathway in the mouse as compared with Xenopus.Our findings disclose another dissimilarity between the mode of regulation of meiosis in amphibian and mammalian oocytes.
A role for p34cdc2 in regulation of Mos stability (45) and Mos translation (46,47) has been suggested for Xenopus oocytes.However, the effect of p34cdc2 on c-mos mRNA polyadenylation is herein demonstrated for the first time.A regulatory step in Mos expression that involves p34cdc2 seems to be shared by both Xenopus and rodent oocytes.However, the absence of a positive feedback loop between MAPK and Mos, demonstrated herein for the rat, apparently represents the different interplay between MPF and MAPK in these two animal models.In Xenopus, p34cdc2 is activated by MAPK.Interference with MAPK activation in these oocytes will be therefore inhibitory for the trans- lation of mos.On the other hand, in the rat, Mos expression does not require an active MAPK since MPF activation in rodents is independent of this kinase (15).
Translational recruitment of stored cytoplasmic mRNA by its polyadenylation, which has been known for several years (48,49), was later shown to be crucial for early development (50)(51)(52).Specifically, c-mos mRNA polyadenylation has been indicated in mouse and Xenopus oocytes (53,56), and its necessity for the completion of oocyte maturation was demonstrated in both these species (53)(54)(55)(56)(57)(58).The determinants involved in cytoplasmic polyadenylation are progressively being identified to include the cytoplasmic polyadenylation element binding protein (CPEB) that is phosphorylated by Eg2, a member of the Aurora family of serine/threonine kinases (59).Furthermore, it has been shown that Eg2 accumulation is induced by a decrease in PKA activity (60).Additionally, Eg2 kinase activity that is undetectable in prophasearrested oocytes is raised in conjunction with p34cdc2 activation (60).Moreover, CPEB phosphorylation that is mediated by p34cdc2 has been reported (61,62).This information has been generated mostly in Xenopus with very little knowledge on mammalian oocytes.Nonetheless, in line with the available information, our findings also suggest a linkage between cytoplasmic polyadenylation, decreased activity of PKA, and activation of p34cdc2.
The presence of the c-mos protooncogene product in mammalian species was demonstrated so far only in murine and bovine oocytes.An early study in the mouse claimed that Mos is present in prophasearrested oocytes (63), while later studies in the same species could not detect Mos in GV oocytes, demonstrating its appearance not earlier then 3 h after GVB (15,17).Similarly, in bovine oocytes, Mos is initially detected at around 4 h after reinitiation of meiosis (64,65).In agreement with these later findings, our study demonstrates that Mos is undetectable in meiotically arrested rat oocytes being initially detected at 6 h after reinitiation of meiosis (2-3 h after GVB).In Xenopus, unlike mammalian oocytes, Mos accumulates before GVB (9), with MAPK activation slightly lagging behind Mos expression (66).Accumulation of Mos in rodents that is delayed until after GVB also corresponds temporally with the late kinetics of MAPK activation demonstrated previously in mouse (17) and confirmed herein for the rat.As expected, Mos synthesis and Mos-dependent downstream events are proceeded by c-mos mRNA polyadenylation.The kinetics of c-mos mRNA polyadenylation and its association with specific stages throughout meiosis has been described previously in the mouse (54) and shown herein for rat oocytes.
The relatively extended time interval that elapses between the decrease in intraoocyte cAMP concentrations and mos translation may raise some concerns in regard to the direct correlation between these two events.By transiently exposing rat oocytes to a (Bu) 2 cAMP/IBMX-free medium followed by their transfer into medium containing the above cAMP elevating agents, we determine herein the exact time point of the cAMP-sensitive initial step of mos translation.This experiment revealed that the drop in intraoocyte cAMP that takes place within the first 30 min after the release of the oocytes from the ovarian follicle is still insufficient to allow mos translation.However, at 45 min, cAMP concentrations are no longer inhibitory and the translation of mos has been initiated.In terms of Mos expression this moment represents a "point of no return" at which any elevation of cAMP is no longer effective.In a previous study, we showed that at 45 min after isolation cAMP concentrations in our rat oocytes drop to 0.7 fmol (28).These findings combined with our present results suggest that these cAMP concentrations can be defined as the threshold requirements for inhibition of mos translation.Collectively, our results provide clear evidence that Mos translation is tightly correlated to the oocyte maturation-associated drop in cAMP and that a 5-h interval indeed elapses between this initial step and c-mos mRNA polyadenylation.This period is apparently used for the completion of a complex cascade of events that may include Eg2 phosphorylation and cytoplasmic polyadenylation element (CPEB) activation (59), which is followed by the cleavage of the polyadenylation-specific factor, which tethers the poly (A) polymerase to the mRNA, capacitating the elongation of the mRNA poly (A) tail (67).The sequence of events that has been described for Xenopus may be relevant, at least in part, for mammalian oocytes.
In summary, in this report we show a tight correlation between the maturation-associated drop in intraoocyte concentrations of cAMP and mos translation.We specifically demonstrate that the translation of mos in rat oocytes is negatively regulated by a PKA-mediated cAMP action that inhibits c-mos mRNA polyadenylation.Cap ribose methylation may be involved in Mos synthesis.MPF, but not MAPK, activation is required for mos translation.These findings, which represent the first demonstration of the control of Mos expression in a mammalian species, depict clear differences in this respect between mammalian and amphibian oocytes.

Oocytes Recovery and Culture
The physiological trigger for reinitiation of meiosis in mammalian oocytes is provided by the preovulatory surge of LH.However, when the oocytes are removed from the ovarian follicles and placed in culture, meiosis resumption occurs spontaneously in the absence of gonadotropins.The ability to resume meiosis in vitro is acquired progressively during oocyte growth.Accordingly, oocytes that are incompetent and competent to resume meiosis are referred to as growing and fully grown, respectively (68).Growing oocytes were isolated from the ovaries of 18-d-old Wistar female rats.Fully grown oocytes were recovered from 25-d-old rats, injected sc with 10 IU PMSG (Chrono-gest Intervest, Oss, The Netherlands) 48 h earlier.Postovulatory oocytes were isolated from 26-dold PMSG (10 IU)-primed rats 18 h after injection with 5 IU human CG (hCG, Pregnyl, Organon, Oss, The Netherlands).Preovulatory cumulus-oocyte complexes were isolated by recovering ovaries into Leibovitz L-15 tissue culture medium containing 10% FBS.The cumulus cells were then removed in acidic L-15 medium (pH 6.0) to obtain cumulus-free oocytes.Postovulatory oocytes were recovered from the oviductal ampulla into L-15 tissue culture medium containing hyaluronidase (1 mg/ml, Sigma) for removal of the cumulus cells.The oocytes were analyzed either immediately after their isolation or after the indicated times of incubation in a 37 C humidified incubator that allowed them to resume meiosis spontaneously in vitro.The oocytes were morphologically examined by differential interference contrast microscopy (Carl Zeiss, Axiovert 35, Oberkochen, Germany).In the presence of GV the oocytes were classified as meiotically arrested.Reinitiation of meiosis was indicated by GVB and progression through MI to reach MII by the formation of PBI.The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory animals (National Research Council, National Academy of Science, Bethesda, MD).

Analysis of c-mos mRNA Levels
The c-mos mRNA was detected by RT-PCR.For this purpose, total oocyte RNA was extracted by the acid-guanidiumphenol-chloroform method (69) and reverse transcribed using specific primers followed by PCR amplification.RT reaction contained 50 U of Moloney murine leukemia virus-reverse transcriptase, 200 M deoxynucleotide triphosphates (dNTPs), 6.5 mM MgCl 2 , 20 U of ribonuclease inhibitor, 500 mg Oligo(dT), and 1.5ϫ PCR buffer (Promega Corp., Madison, WI).The reaction was carried out at 37 C for 2 h.Fragments of the reverse transcribed c-mos cDNA were amplified using a labeled nucleotide ([␣-32 P]dCTP, Amersham Pharmacia Biotech), and the following pairs of primers were employed: 5Ј-GCACCACGA-CAACATAATCC-3Ј and 5Ј-CAGCCGAAGTCACTTATCTTAC-3Ј for c-mos.
A fragment of the ribosomal S16 cDNA, which served as our internal control, was amplified simultaneously using the two following primers: 5Ј-CGTTCACCTTGATGAGCCC-ATT-3Ј and 5Ј-TCCAAGGGTCCGCTGCAGTC-3Ј.PCR reactions were further performed in the same RT-test tube that finally contained 250 ng of each primer, 200 M dNTP, 2.5 mM MgCl 2 , 2 Ci [␣-32 P]dCTP, 1ϫ PCR buffer (Promega Corp.), and 2.5 U Taq polymerase.Thirty and 28 cycles for S-16 and c-mos, respectively, were performed after a 2-min incubation at 94 C as follows: 94 C for 30 sec; 60 C for 30 sec, and 72 C for 1 min.This was followed by a final extension for 5 min at 72 C. The radioactive products were electrophoresed on 5% nondenaturing polyacrylamide gel in 0.5ϫ Tris-borate-EDTA buffer.Gels were dried and radioactivity was determined by exposure to x-ray film.Quantitation and comparison of the different samples on the autoradiograms was performed by densitometric analysis using the 420oe densitometer (PDI, Huntington Station, NY) followed by normalization according to the internal standard.

Analysis of c-mos mRNA Polyadenylation
c-mos mRNA polyadenylation was detected using the PCR as described by Sall'es and Strickland (31).Briefly, total oocyte RNA was extracted by the acid-guanidium-phenolchloroform method (69).An aliquot of RNA in H 2 O (equivalent to 100-150 oocytes) was heat denatured (65 C for 5 min) in a 7 l volume in the presence of 20 ng of phosphorylated oligo(dt) (Promega Corp.) and placed directly at 42 C. Prewarmed mastermix [13 l containing 4 l of 5ϫ Superscript RNase H Ϫ RT buffer (Life Technologies, Inc.); 2 l of 0.1 M DTT; 1 l of 10 mM dNTPs; 1 l of 10 mM ATP; 4 l of H 2 O; and 1 l of 10 U/l T4 DNA ligase (Promega Corp.) was added, and the samples incubated at 42 C for 30 min.Subsequently, 1 l of oligo(dt)-anchor (200 ng/l, 5Ј-GCG-AGCTCCGCGGCCGCGT12-3Ј) was added at 42 C and the reaction was transferred to a 12-C water bath.After a 2-h incubation, the samples were transferred back to 42 C, 2 l (200 U/l) SuperScript RNase H Ϫ RT was added (Life Technologies, Inc.), and reverse transcription was performed for 1 h.cDNAs were diluted to six oocytes/l followed by 30 min incubation at 70 C to inactivate the RT and the ligase.For PCR amplification, 1 l of cDNAs was added to a standard 50 l PCR reaction spiked with 0.5 l of [␣-32 P]dATP and con- taining 25 pmol each of mos mRNA specific primer (5Ј-GCACCACGACAACATAATCC-3Ј) and the Oligo(dT)-anchor (amplification conditions: 93 C for 5 min; 30 cycles at 93 C for 30 sec; 62 C for 1 min; 72 C for 1 min; with a final extension of 7 min at 72 C).After amplification, PCR products were ethanol-precipitated with 2.5 M ammonium acetate to remove unincorporated label.To confirm the specificity of the amplification and that the size increase of the PCR fragments was attributable to elongation of the 3Ј-end of the mRNA, samples were digested with XmnI (Promega Corp.) and showed a heterogeneous 3Ј-end.The radioactive products were electrophoresed on 5% nondenaturing polyacrylamide gel in 0.5ϫ Tris-borate-EDTA buffer.Gels were dried and radioactivity was determined by exposure to x-ray film overnight.

PKA Phosphorylation Assay
The activity of PKA was tested in a cell-free reaction system using vitronectin as a substrate.The assay was conducted as described previously (71).Rat granulosa cells were lysed in buffer containing 50 mM ␤-glycerophosphate, 1 mM NaF, 1.5 mM EGTA, 1% Nonident P-40, 1 mM EDTA, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin, and 1 mM DTT. Phosphorylation was carried out in a final volume of 50 l containing different doses of inhibitor, 50 mM HEPES (pH 7.3), 10 mM MgCl 2 , 0.5 g vitronectin.Phosphorylation was initiated by adding 10 M [␥-32 P]ATP (6 Ci/mmol).The reaction was allowed to proceed 20 min at room temperature and arrested by addition of 5ϫ sample buffer (250 mM Tris, pH 6.8; 10% SDS; 50% glycerol; 0.006% bromophenol blue; 125 mM DTT) and boiling for 5 min at 100 C. The samples were subjected to SDS-PAGE.Gels were dried and radioactivity was determined by their exposure to x-ray film for 3 h at room temperature.The assay was conducted in the absence and presence of cAMP (10 mM) as well as the specific PKA inhibitor, PKI (30 M) to determine its specificity and to establish the cAMP dependency of this phosphorylation.

Fig. 1 .
Fig. 1.The Pattern of Mos Expression and MAPK Activation Throughout Spontaneous Oocyte Maturation A, Kinetics of spontaneous maturation in rat oocytes resuming meiosis.Oocytes were recovered from the ovarian follicles and incubated as described in Materials and Methods.The oocytes were microscopically examined at the indicated time points for GVB and PBI formation.A total number of at least 335 oocytes was examined for each time point in three individual experiments.The results of three repetitions are presented along with their SEs.B, RT-PCR analysis of c-mos mRNA expression in rat oocytes resuming meiosis.RNA was extracted from oocytes (100/lane) at the indicated time points of their incubation with and without (Bu) 2 cAMP (2 mM) and IBMX (20 M). c-mos mRNA was analyzed using radioactive RT-PCR as described in Materials and Methods.The results of one representative experiment of four repetitions are presented.C, The pattern of Mos accumulation in rat oocytes resuming meiosis.Spontaneously maturing rat oocytes (500/lane) were extracted at the indicated time points after reinitiation of meiosis.The extracts were separated and immunobloted with anti-Mos antibodies.Inset, Depletion of the anti-Mos antibodies by preincubation with a p41Mos synthetic peptide completely eliminated the signal.The results of one representative experiment of four repetitions are presented.D, The pattern of MAPK activation throughout meiosis.The oocytes mentioned in panel C (50/lane) were extracted at the indicated time points after reinitiation of meiosis.Postovulatory oocytes (PO) that served as a control for the 24-h time point were also included.The extracts were electrophoretically separated and immunoblotted with anti-P-MAPK and anti-total MAPK antibodies.Western blot analysis for the presence of Mos was performed as described for panel C. Upper panel, Autoradiogram of one representative experiment of five repetitions.Lower panel, Means Ϯ SEs of quantitative analysis of these five experiments.

Fig. 5 .
Fig. 5. Reversal of cAMP Action by a PKA InhibitorA, Sensitivity of PKA to 4-cyano-3-methylisoquinoline (4C3M).PKA sensitivity to 4C3M was analyzed in the presence of increasing concentrations of 4C3M using rat granulosa cell lysates, and phosphorylation assay was conducted as described in Materials and Methods.The results of one representative experiment of three repetitions are presented.B, The effect of 4C3M on Mos accumulation.Oocytes (500/lane) were incubated for 8 h in the presence and absence of (Bu) 2 cAMP (2 mM), IBMX (20 M), and 4C3M (200 nM) in the culture medium.Western blot analysis was performed as described for Fig.1C.The results of one representative experiment of three repetitions are presented.

Fig. 6 .
Fig. 6. c-mos mRNA Polyadenylation Throughout Oocyte Maturation: Inhibition by cAMP and Reversal of Inhibition by a PKA Inhibitor A, Poly(A) tail analysis (PAT) of c-mos mRNA throughout meiosis.RNA was extracted from oocytes at the indicated time points of incubation in the presence or absence of (Bu) 2 cAMP (2 mM), IBMX (20 M), and 4C3M (200 nM).c-mos mRNA was subjected to PAT-RT as described in Materials and Methods.Six oocytes equivalent of PAT-cDNAs was amplified per time point of treatment as mentioned in Materials and Methods.The results of one representative experiment of five repetitions are presented.B, The effect of the methyltransferase inhibitor, SIBA, on Mos expression.Oocytes were incubated in the presence or absence of SIBA (0.8 mM) for 8 h.Western blot analysis was performed as described for Fig. 1C.The results of one representative experiment of three repetitions are presented.

Fig. 7 .
Fig. 7.The Effect of Roscovitine on c-mos mRNA Polyadenylation RNA was extracted from oocytes after 8 h of incubation in the presence or absence of roscovitine (100 M).Poly(A) tail analysis (PAT) of c-mos mRNA was performed as described for Fig. 5A.The results of one representative experiment of three repetitions are presented.

Fig. 8 .
Fig. 8. Mos Expression Is Independent of an Active MAPKA, The effect of PD 98059 on MAPK activation.Oocytes were incubated for 8 h in the presence and absence of the MEK inhibitor PD 98059 (50 M) in the culture medium.The oocytes (50/lane) were extracted after 8 h of incubation.The extracts were electrophoretically separated and immunobloted with anti-P-MAPK and anti-total MAPK antibodies as described for Fig.1C.The results of one representative experiment of three repetitions are presented.B, The effect of PD 98059 on Mos accumulation.Oocytes were incubated for 8 h in the presence and absence of PD 98059 (50 M) in the culture medium.Western blot analysis was performed as described for Fig.1C.The results of one representative experiment of three repetitions are presented.C, The effect of the MEK inhibitor U0126 on MAPK activation.Oocytes were incubated for 8 h in the presence and absence of U0126 (8 M) in the culture medium.The oocytes (50/lane) were extracted after 8 h of incubation.The extracts were electrophoretically separated and immunoblotted with anti-P-MAPK and anti-total MAPK antibodies as described for Fig.1C.The results of one representative experiment of three repetitions are presented.D, The effect of U0126 on Mos accumulation.Oocytes were incubated for 8 h in the presence and absence of U0126 (8 M) in the culture medium.Western blot analysis was performed as described for Fig.1C.The results of one representative experiment of three repetitions are presented.