cAMP-Dependent PKA Negatively Regulates Polyadenylation of c-mos mRNA in Rat Oocytes
Shlomi Lazar,
Dalia Galiani and
Nava Dekel1
Department of Biological Regulation, The Weizmann Institute of
Science, Rehovot 76100, Israel
Address all correspondence and requests for reprints to: Professor Nava Dekel, Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail:
nava.dekel{at}weizmann.ac.il
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ABSTRACT
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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.
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INTRODUCTION
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MEIOSIS 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
cAMP-dependent 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 PKA- mediated 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.
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RESULTS
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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 26 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.

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Figure 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)2cAMP (2 mM) and IBMX (20
µM). 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 ± SEs of quantitative analysis
of these five experiments.
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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)2cAMP (2 mM) together
with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (20
µM), 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 MII-arrested 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 µM), 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 µg/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.

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Figure 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 µM) 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 µg/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.
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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)2cAMP (2 mM) and the cAMP
phosphodiesterase inhibitor, IBMX (20 µM). 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
intraoocyte concentration of cAMP also resulted in inhibition of MAPK
activation (Fig. 3B
).

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Figure 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)2cAMP (2 mM) and IBMX (20 µM)
in the culture medium. When indicated, removal of (Bu)2cAMP
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.
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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)2cAMP/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.

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Figure 4. Timing of cAMP-Sensitive Initial Step for Mos
Expression
Freshly isolated oocytes (500/lane) were exposed to
(Bu)2cAMP/IBMX-free medium for varying periods of time. The
oocytes were then transferred into medium containing
(Bu)2cAMP (2 mM) and IBMX (20 µM)
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.
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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
).

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Figure 5. Reversal of cAMP Action by a PKA Inhibitor
A, 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)2cAMP (2 mM), IBMX
(20 µM), 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.
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The translation of mos in oocytes is mediated by its polyadenylation
(30). To explore the mode of regulation 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)2cAMP
and IBMX. However, the inhibitory effect of
(Bu)2cAMP on c-mos mRNA
polyadenylation was effectively reversed by the PKA inhibitor, 4C3M
(200 nM, Fig. 6A
).

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Figure 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)2cAMP (2
mM), IBMX (20 µM), 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.
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Cytoplasmic polyadenylation in Xenopus oocytes stimulates
cap ribose methylation (32). A more recent study further
demonstrated that cap ribose methylation 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
µM, 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.

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Figure 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 µM). 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.
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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.
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DISCUSSION
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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 regulated 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 translation 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 prophase-arrested 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 prophase-
arrested 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 (23 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)2cAMP/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.
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MATERIALS AND METHODS
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Reagents and Antibodies
Leibovitzs L-15 tissue culture medium was purchased from
Life Technologies, Inc. (Paisley, UK). Antibiotics were
purchased from Bio-Lab Ltd. (Jerusalem, Israel). MG132
(Z-leu-leu-CHO), 4-cyano-3-methylisoquinoline, PKI, and
U0126 were purchased from Calbiochem (La Jolla, CA). IBMX,
(Bu)2cAMP, cycloheximide, SIBA, Nonident P-40, ß-glycerophosphate,
phenylmethylsulfonylfluoride (PMSF), leupeptin, aprotonin, DTT, and FBS
were purchased from Sigma (St. Louis, MO). Affinity
purified polyclonal goat anti-Mos antibody was purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Polyclonal rabbit anti-MAPK and monoclonal mouse anti P-MAPK were
kindly provided by Prof. Rony Seger, from The Weizmann Institute of
Science (Rehovot, Israel). Donkey antigoat, goat antimouse, and goat
antirabbit peroxidase-conjugated antibodies were purchased from
Jackson ImmunoResearch Laboratories, Inc. (West Grove,
PA). [
-32P]dCTP,
[
-32P]dATP,
[
-32P]ATP, and enhanced chemiluminescence
Western blotting detection reagents were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). PD 98059 was
purchased from BIOMOL Research Laboratories, Inc.
(Plymouth Meeting, PA). Roscovitine was purchased from
Calbiochem (La Jolla, CA). Vitronectin was kindly
provided by Prof. Shmuel Shaltiel, from The Weizmann Institute of
Science (Rehovot, Israel).
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-d-old 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-guanidium-phenol-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 µM
deoxynucleotide triphosphates (dNTPs), 6.5 mM
MgCl2, 20 U of ribonuclease inhibitor, 500
mg Oligo(dT), and 1.5x 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
([
-32P]dCTP, Amersham Pharmacia Biotech), and the following pairs of primers were employed:
5'-GCACCACGACAACATAATCC-3' and 5'-CAGCCGAAGTCACTTATCTTAC-3' for
c-mos.
A fragment of the ribosomal S16 cDNA, which served as our internal
control, was amplified simultaneously using the two following
primers: 5'-CGTTCACCTTGATGAGCCC- ATT-3' and 5'-TCCAAGGGTCCGCTGCAGTC-3'.
PCR reactions were further performed in the same RT-test tube that
finally contained 250 ng of each primer, 200 µM dNTP, 2.5
mM MgCl2, 2 µCi
[
-32P]dCTP, 1x 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 0.5x 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 Salles and Strickland (31). Briefly,
total oocyte RNA was extracted by the acid-guanidium-phenol-chloroform
method (69). An aliquot of RNA in
H2O (equivalent to 100150 oocytes) was heat
denatured (65 C for 5 min) in a 7 µl volume in the presence of 20 ng
of phosphorylated oligo(dt) (Promega Corp.) and placed
directly at 42 C. Prewarmed mastermix [13 µl containing 4 µl of
5x Superscript RNase H- RT buffer (Life Technologies, Inc.); 2 µl of 0.1 M DTT;
1 µl of 10 mM dNTPs; 1 µl of 10
mM ATP; 4 µl of H2O; and
1 µl of 10 U/µl T4 DNA ligase (Promega Corp.) was
added, and the samples incubated at 42 C for 30 min. Subsequently, 1
µl of oligo(dt)-anchor (200 ng/µl, 5'-GCGAGCTCCGCGGCCGCGT12-3')
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 µl (200 U/µl) SuperScript RNase H- RT
was added (Life Technologies, Inc.), and reverse
transcription was performed for 1 h. cDNAs were diluted to six
oocytes/µl followed by 30 min incubation at 70 C to inactivate the RT
and the ligase. For PCR amplification, 1 µl of cDNAs was added to a
standard 50 µl PCR reaction spiked with 0.5 µl of
[
-32P]dATP and containing 25 pmol each of
mos mRNA specific primer (5'-GCACCACGACAACATAATCC-3') 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 3'-end of the mRNA, samples were
digested with XmnI (Promega Corp.) and showed a
heterogeneous 3'-end. The radioactive products were electrophoresed on
5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA
buffer. Gels were dried and radioactivity was determined by
exposure to x-ray film overnight.
Western Blot Analysis
Samples of 500 oocytes were extracted in a lysis buffer
(ß-glycerophosphate, 50 mM; EGTA, 1.5 mM;
EDTA, 1 mM; sodium-orthovanadate, 0.1 mM;
benzamidine, 1 mM; aprotonin, 10 µg/ml; leupeptin, 10
µg/ml; pepstatin A, 2 µg/ml; DTT, 1 mM; PMSF, 1
mM), and Laemmli sample buffer (70) was added.
The samples were boiled and loaded on 12% SDS-PAGE, followed by their
transfer to a nitrocellulose membrane. After blocking with Tween 20 +
Tris-buffered saline containing 10% skimmed milk, the membranes
were incubated with the relevant antibodies. The following antibodies
were used: affinity purified polyclonal goat anti-Mos antibody (1:500
dilution), rabbit anti-MAPK (1:5,000 dilution) and mouse
antiphosphorylated MAPK (P-MAPK, 1:5,000 dilution). Donkey antigoat,
goat antirabbit, and goat antimouse horseradish peroxidase-conjugated
second antibodies were used (1:5,000 dilution) and immunoreactive bands
were detected using enhanced chemiluminescence reagents. Quantitation
of the autoradiograms was performed by densitometric analysis as
described previously in this section.
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 ß-glycerophosphate, 1
mM NaF, 1.5 mM EGTA, 1%
Nonident P-40, 1 mM EDTA, 1
mM PMSF, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 1 mM DTT. Phosphorylation
was carried out in a final volume of 50 µl containing different doses
of inhibitor, 50 mM HEPES (pH 7.3), 10
mM MgCl2, 0.5 µg
vitronectin. Phosphorylation was initiated by adding 10
µM [
-32P]ATP (6
Ci/mmol). The reaction was allowed to proceed 20 min at room
temperature and arrested by addition of 5x 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 µM)
to determine its specificity and to establish the cAMP dependency of
this phosphorylation.
 |
FOOTNOTES
|
---|
This work was partially supported by a grant from the Maria Zondek
Hormone Research Fund (N.D.).
1 Incumbent of the Phillip M. Klutznick professorial chair of
Developmental Biology. 
Abbreviations: CPEB, Cytoplasmic polyadenylation element
binding protein; dNTPs, deoxynucleotide triphosphates; DTT,
dithiothreitol; GVB, germinal vesicle breakdown; IBMX,
isobutylmethylxanthine; MEK, MAPK kinase; MI and MII, first and
second metaphase, respectively; MPF, maturation- promoting
factor; PBI, first polar body; PMSF, phenylmethylsulfonylfluoride;
SIBA, S-isobutyl-thioadenosine.
Received for publication March 23, 2001.
Accepted for publication October 16, 2001.
 |
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