Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print December 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.024737
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BIOLOGY OF REPRODUCTION 70, 1178–1187 (2004)
DOI: 10.1095/biolreprod.103.024737
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Protein Kinase C and Mitogen-Activated Protein Kinase Cascade in Mouse Cumulus Cells: Cross Talk and Effect on Meiotic Resumption of Oocyte1

Heng-Yu Fan3,4, Li-Jun Huo3, Da-Yuan Chen3, Heide Schatten5, and Qing-Yuan Sun2,3

State Key Laboratory of Reproductive Biology,3 Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China Department of Molecular Biology,4 University of Texas Southwestern Medical Center, Dallas, Texas 75390 Department of Veterinary Pathobiology,5 University of Missouri-Columbia, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.

gamete biology, kinases, meiosis, oocyte development, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fully grown mammalian oocytes are arrested at prophase of the first meiotic division within the ovarian follicles. In vivo, meiotic resumption of oocytes occurs in response to the preovulatory surge of gonadotropins, especially LH. The mechanisms by which meiosis is inhibited or induced in the ovarian follicular environment are not well understood. The follicular fluid contains purines such as hypoxanthine (HX) that are considered to be important inhibitory substances of oocyte maturation in mammalian species such as mouse [1], rat [2], pig [3], and sheep [4]. It has been reported that cumulus cells can produce a gonadotropin-dependent, positive stimulus to resume meiotic resumption [5]. Cumulus cells may promote the meiotic maturation of oocytes in a paracrine pathway by secreting a meiosis-activating substance when stimulated by gonadotropin [6]. Furthermore, gap junctions connecting the oocytes with the surrounding cumulus cells within the follicle allow the transfer of small molecules from somatic cells to the germ cell [7].

Basically, two models have been employed in studying the rodent oocyte meiotic resumption in vitro: the spontaneous meiosis model and the induced meiosis model. Mouse and rat oocytes resume meiosis spontaneously when they are released from the inhibitory environment of follicles. Cumulus cell-enclosed oocytes (CEOs) also mature in vitro upon stimulation by gonadotropin or epidermal growth factor (EGF) when the spontaneous maturation is prevented by meiotic inhibitors, such as hypoxanthine and cAMP-elevating agents [8]. In these two models, different mechanisms are employed in regulating the progression of the meiotic cell cycle. The former is thought to be a relatively passive response to the artificial removal from a meiotic-inhibiting environment of the follicles, whereas the latter is more active, presumably requiring gonadotropin-triggered production of a meiosis-inducing stimulus. Importantly, these ligands act through mediation by cumulus cells, as they fail to promote the same effect on denuded oocytes (DOs).

Several ubiquitously expressed protein kinases—including protein kinase A (PKA) [9, 10]; protein kinase C (PKC) [11, 12]; calcium/calmodulin-dependent protein kinase II (CaMKII) [13, 14]; and mitogen-activated protein kinase (MAPK) [15–17]—are involved in the regulation of meiotic maturation in both oocytes and cumulus cells. Intriguingly, their effects on meiotic resumption sometimes appear to be controversial in the spontaneous and the induced oocyte maturation models. Activation of type I PKA by elevated cAMP level in the oocyte prevents spontaneous meiotic resumption, whereas gonadotropin-stimulated activation of type II PKA within the cumulus cells leads to meiotic induction in CEOs [18]. A similar phenomenon was also observed while studying the roles of PKC in meiotic resumption of mammalian oocytes. Results from our laboratory and others indicated that PKC activators, such as phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), significantly inhibited the spontaneous meiotic resumption in mouse [11], rat [12], and pig [19] oocytes. At the same time, activation of MAPK cascade was also inhibited or retarded in these oocytes, although the same reagents overcame the prophase arrest maintained by HX in mouse [1] and pig CEOs [3]. Except for its effects in mammalian oocytes, PKC is also involved in the meiotic induction of oocytes in low vertebrates and invertebrate species. Germinal vesicle (GV)-stage arrested Xenopus oocytes can be induced to undergo germinal vesicle breakdown (GVBD) by progesterone and insulin. Microinjection of the PKC {delta} catalytic domain also induced the GVBD of Xenopus oocytes and enhanced the insulin-induced but not progesterone-induced maturation [20]. PKC activation induced meiotic maturation of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment [21]. In frog oocytes, diacylglycerol (DG) levels can increase transiently after exposure to insulin-like growth factor, insulin, or progesterone [22]. Microinjection of v-ras protein into frog oocytes also increased DG turnover and caused GVBD [23]. Activation of PKC in Caetopteros oocytes leads to maturation-promoting factor (MPF) activation and GVBD. Diacylglycerol, the second signal transducer, facilitates PKC activation, increases as GVBD is induced, and then decreases dramatically before fertilization [24]. However, involvement of MAPK cascade in GVBD induced by PKC activators has not been carefully studied in any species.

MAPK, which is also termed extracellular-regulated kinase (ERK), is another family of Ser/Thr protein kinases that play a pivotal role in meiosis (for review, see [25–27]). Accumulating evidence suggests that MAPK activity is not required for the spontaneous meiotic resumption of mouse and rat oocytes, since it is activated 2 h after spontaneous GVBD [12, 28, 29]. In denuded mouse oocytes, GVBD occurred normally when MAPK activity was inhibited by the MEK inhibitor U0126 [30]. Furthermore, in c-mos knockout mice, oocytes could also resume meiosis, although MAPK failed to be activated in these cells [31, 32]. Studies in farm animals revealed the same results as in rodents. Microinjection of MKP-1 mRNA, which encodes a specific MAPK phosphatase, into GV-stage bovine oocytes could not prevent the meiotic resumption [33]. Microinjection of porcine c-mos antisense RNA into pig oocytes failed to arrest the cells at the GV stage [34]. We also reported that the spontaneous meiotic resumption of pig DOs could occur normally when MAPK phosphorylation was strongly inhibited by U0126 [17]. However, MAPK activity is necessary for the gonadotropin-induced meiotic resumption of mouse and pig oocytes. MEK inhibitors, PD98059 and U0126, prevent FSH-induced GVBD in mouse and pig CEOs by inhibiting the MAPK activation in cumulus cells [15–17]. In GV-stage arrested Xenopus oocytes, accumulation of MOS above a threshold level activates MAPK pathway [35], which leads to MPF activation, most likely through activation of p90rsk. Activated p90rsk inactivates Myt1, a negative regulating kinase of MPF, thereby promoting activation of MPF and entry into meiosis I [36]. MAPK cascade may also facilitate the activation of MPF by stimulating accumulation of cyclin B, the regulatory subunit of MPF [26]. But in mammalian oocytes, the pathway by which MAPK leading to MPF activation and GVBD is less definitive as that in Xenopus oocytes. Despite these previous studies, the functions and interactions of PKC and MAPK cascade in mammalian cumulus cells are largely unknown. It appears that in cumulus cells MAPK is not activated by the de novo synthesis of MOS, the specific MEK kinase in germ cells, because MAPK could be activated in the cumulus cells derived from c-mos knockout mice after FSH stimulation [16]. The pathway by which PKC is involved in FSH-induced meiotic resumption of CEOs has never been investigated. The present study aims to elucidate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in inducing meiotic resumption in mouse CEOs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Stock solutions of PMA (1.62 mM); 4{alpha}-phorbol 12, 13-didecanoate (4{alpha}-PDD, 1.62 mM); OAG (100 mM; Calbiochem, La Jolla, CA); calphostin C (Cal, 1 mM); chelerythrine (5 mM; Calbiochem); cycloheximide (CHX, 2 mg/ml); cordycepin (50 mM); and U0126 (15 mM) were prepared in dimethyl sulfoxide (DMSO) and stored frozen at -20°C until use. Prior to use, they were diluted with culture medium. Stock solution of FSH (100 IU/ml) was prepared in M199 and stored at -20°C. All chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted.

Oocyte Collection and Culture

Oocytes were collected from Kunming strain mice. Animal care and handling were conducted in accordance with policies on the care and use of animals promulgated by the ethical committee of the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. Fully grown germinal vesicle (GV)-intact oocytes were collected from ovaries of 21- to 24-day-old mice 44–46 h after the females were intraperitoneally injected with 5 IU eCG. Cumulus-enclosed and GV-intact oocytes were released from antral follicles by puncturing the follicles with a needle in culture medium. The culture medium used for this study was M199 medium containing 4 mM hypoxanthine; 0.23 mM sodium pyruvate; 2 mM glutamine; 3 mg/ml lyophilized crystallized BSA (Calbiochem, CA); 75 µg/ml potassium penicillin G; and 50 µg/ml streptomycin sulphate. This medium is termed "HX medium". The above-described medium without hypoxanthine was designated as HX-free medium. In each experiment, a group of 30 oocytes was cultured in a 100 µl drop covered with paraffin oil in a 3.5-mm culture dish. All cultures were carried out at 37°C in a humidified atmosphere of 5% CO2.

Western Blot Analysis

For detection of p90rsk and active ERK1/2, proteins from 30 oocytes or cumulus cells of 30 COCs were collected in double strength SDS sample buffer and heated to 100°C for 4 min. After cooling on ice and centrifuging at 12 000 x g for 3 min, samples were frozen at -20°C until use. The total proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel for 20 min at 90 V and 4.5 h at 110 V, respectively, and then electrophoretically transferred onto nitrocellulose membrane for 2.5 h, 200 mA, at 4°C. The membrane was then blocked overnight at 4°C in TBST buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.4) containing 5% low-fat milk. To detect both p90rsk and active ERK1/2, blots were cut in two parts containing the proteins above and below the 68 kDa marker and incubated separately for 2 h in TBST with 1:300 polyclonal rabbit anti-mouse p90rsk antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for the upper part of the membrane and with 1:500 mouse anti-p-ERK1/2 antibody (Santa Cruz Biotechnology) for the lower part. After three washes of 10 min each in TBST, the upper and lower parts of the membrane were incubated for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated rabbit anti-mouse IgG diluted 1:1000 in TBST, respectively. The membranes were washed three times in TBST and then processed using the enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK).

For reprobing of total ERK2, the lower part of the membrane was washed in stripping buffer (100 mM ß-mercaptoethanol, 20% SDS, 62.5 mM Tris pH 6.7) to strip off bound antibody after enhanced chemiluminescence detection at 50°C for 30 min. The membrane was reprobed with polyclonal rabbit anti-ERK2 antibody (Santa Cruz Biotechnology) diluted 1:300, incubated with HRP-labeled goat anti-rabbit IgG, and finally processed as described above.

Oocyte Assessment and Statistical Analysis

Oocytes were cultured for various times, denuded of their cumulus cells by repeated pipetting, and morphologically assessed for the presence of the GV using an inverted microscope equipped with differential interference contrast. Oocytes showing signs of degeneration or morphological abnormality were excluded from the statistical analysis. In all experiments the proportion of oocytes with a normal appearance was at least 90%. Each experiment was repeated at least three times with at least 30 oocytes for each experiment in each group. Frequencies of GVBD in various groups were subjected to arc sin transformation and statistically compared by ANOVA followed by the Student-Newman-Keuls test.

Experimental Design

Experiment 1 As a preliminary experiment, CEOs or DOs were cultured in HX medium, HX-free medium, or HX medium containing 100 IU/L FSH. The GVBD rate was recorded at 16 h after culture. Samples of oocytes or cumulus cells were collected for Western blot analysis of MAPK phosphorylation after culture.

Experiment 2 The effects of PKC activators and inhibitors on meiotic resumption and MAPK phosphorylation in CEOs or DOs were studied. The CEOs or DOs were cultured in HX medium containing PKC activator PMA (16.2 nM) or OAG (50 µM), PKC inhibitor calphostin C (5 µM), or 16.2 nM PMA plus 5 µM calphostin C for 16 h, and the oocytes were then collected for GVBD observation and Western blot analysis. Kinetics of meiotic resumption and MAPK activation in CEOs was also recorded. CEOs were cultured in HX medium with or without 16.2 nM PMA for up to 24 h. GVBD was scored and MAPK phosphorylation was tested at 4-h intervals. As the control, PMA was substituted by its biologically inactive analog 4{alpha}-PDD at the same concentration.

Experiment 3 Possible regulation of PKC on MAPK activity in cumulus cells was tested. Mouse CEOs were cultured in HX medium containing 16.2 nM PMA or 16.2 nM PMA plus 5 µM calphostin C. Cumulus cells were collected at 0, 2, 4, 8, 12, or 16 h of drug treatment for Western blot analysis. As the control, CEOs were cultured in drug-free HX medium.

Experiment 4 Possible involvement of PKC in FSH-induced meiotic resumption in mouse CEOs was studied. CEOs were cultured in HX medium containing 100 IU/L FSH plus 5 µM calphostin C or 100 IU/L FSH plus 5 µM chelerythrine. GVBD rate was recorded at 16 h of culture. Samples of both the oocytes and cumulus cells were collected for detection of MAPK phosphorylation.

Experiment 5 MEK inhibitor U0126 was used to determine the requirement of MAPK cascade in FSH- or PMA-induced meiotic resumption in mouse CEOs. CEOs were cultured in HX medium containing 100 IU FSH or 16.2 nM PMA, plus 10 µM U0126 each. GVBD and MAPK phosphorylation in oocytes were investigated at 16 h of culture. MAPK phosphorylation in cumulus cells was detected at 4 and 8 h of culture.

Experiment 6 To determine if the cumulus cells exert their effect on the meiotic resumption of oocytes by secreting a paracrine factor or through gap junctions after PKC activation, the cumulus cells and oocytes of COCs were separated at the beginning of culture, and the two types of cells (cumulus cells and denuded oocytes) were cultured in the same drop. Media used for coculture were HX medium containing 16.2 nM PMA and HX medium with 16.2 nM PMA and 10 µM U0126. GVBD and MAPK phosphorylation in DOs were tested 16 h after coculture. MAPK activation in isolated cumulus cells were tested at 4 h of coculture.

Experiment 7 The requirement for protein synthesis in FSH- and PMA-induced meiotic resumption was investigated. CEOs were incubated with HX medium containing 100 IU/L FSH plus 5 µg/ml eukaryotic protein synthesis inhibitor CHX or 50 µM cytoplasmic polyadenylation inhibitor cordycepin. In another group, CEOs were cultured with 16.2 nM PMA plus 5 µg/ml CHX or 50 µM cordycepin. The effect of CHX on spontaneous GVBD of DOs was also studied. DOs were cultured in HX-free medium containing 5 µg/ml CHX. In all groups, nuclear status was examined 16 h after incubation. Cumulus cells were collected at 4 h of incubation for Western blot analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FSH Stimulates the Meiotic Resumption and MAPK Activation in CEOs

In the preliminary experiment shown in Figure 1A, we found that a very low proportion of CEOs (28.13%, n = 128) or DOs (12.5%, n = 112) resume meiosis spontaneously in HX medium for at least 16 h, consistent with previous reports [15, 37]. To mimic meiotic resumption in vivo, CEOs were exposed to FSH, as originally described [38]. After 16 h of incubation, treatment with FSH promoted GVBD in 89.75% (n = 78) of CEOs cultured in HX medium.



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  		FIG. 1. Effect of PKC inhibitors (calphostin C and chelerythrine) and MEK inhibitor U0126 on FSH- and PKC-induced meiotic resumption of mouse oocytes. A) Mouse CEOs were cultured for 16 h in HX medium supplemented with or without 100 IU/L FSH to show the inductive effect of FSH on GVBD of oocytes. CEOs were also cultured in FSH-containing HX medium with 5 µM calphostin C, 5 µM chelerythrine, or 10 µM U0126. B) Mouse CEOs were cultured for 16 h in drug-free HX medium or HX medium supplemented with the following drugs: 16.2 nM PMA, 50 µM OAG, 16.2 nM PMA + 5 µM calphostin C, 16.2 nM PMA + 10 µM U0126, 5 µM OAG + 5 µM chelerythrine, or 5 µM OAG + 10 µM U0126. Data are shown as mean percentage of GVBD ± SEM for three independent experiments. Different superscripts denote statistical difference at a P < 0.05 level of significance in the GVBD of oocytes. This statistical analysis is also used in the following figures

Phosphorylation of MAPK was studied in both oocytes and cumulus cells with or without FSH treatments. Furthermore, the activity of p90rsk, the best known target of MAPK in oocytes, was also assessed. The full phosphorylation of p90rsk is MAPK-dependent, and its phosphorylation status is another indicator of MAPK activity in oocytes. As shown in Figure 2A, MAPK and p90rsk were inactive in oocytes just isolated from follicles (lane 1). These two kinases remained inactive when the CEOs were cultured in HX medium for 16 h (lane 2). Phosphorylation of MAPK and p90rsk was detected in CEOs cultured in HX medium containing FSH for 16 h (lane 3). In cumulus cells, most MAPK existed in a dephosphorylated form when just isolated from the follicles (Fig. 2B, lane 1) and kept inactive after 8 h of culture in the HX medium (Fig. 2B, lane 2). Phosphorylation of MAPK was detected in cumulus cells when the CEOs were cultured in HX medium containing FSH for 2, 4, and 8 h with increasing levels of intensity (Fig. 2B, lanes 3–6), indicating that FSH is capable of inducing MAPK activation in cumulus cells. In this experiment, the mobility shift of p90rsk was not detected in cumulus cells with or without FSH treatment.



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  		FIG. 2. Regulation of MAPK/p90rsk activity in mouse COCs during FSH-induced oocyte meiotic resumption. Samples for immunoblotting were collected after evaluation of oocyte maturation status at the end of culture. A total of 30 oocytes or cumulus cells from the same 30 CEOs were loaded onto each lane. Blots were incubated with a polyclonal anti-p90rsk antibody and a polyclonal anti-active MAPK antibody to show p90rsk mobility shift (upper panel) and MAPK activity (middle panel). The same blots were subsequently stripped and reprobed with polyclonal anti-ERK2 antibody (lower panel). The same treatments were applied in all subsequent Western blot figures. A) Oocyte samples collected after the following treatment: oocytes isolated from follicles (lane 1), CEOs cultured for 16 h in HX medium (lane 2), HX medium containing 100 IU/L FSH (lane 3), 100 IU/L FSH + 10 µM U0126 (lane 4), and FSH + 5 µM calphostin C (lane 5). B) Cumulus cell samples were collected after the following treatment: COCs isolated from follicles (lane 1) or cultured for 8 h in HX medium (lane 2); COCs cultured for 2, 4, and 8 h in HX medium containing 100 IU/L FSH (lanes 3–5); COCs cultured for 4 and 8 h in HX medium containing 100 IU/L FSH 10 µM U0126 (lane 6 and 7); and COCs cultured for 4 and 8 h in HX medium containing 100 IU/L FSH 5 µM calphostin C (right panels)

PKC Activators Induce the Meiotic Resumption and MAPK Activation in COCs

To prevent the nonspecific toxic effect of chemical reagents on the cultured cells, two structurally unrelated PKC activators, PMA and OAG, were used in the following experiments. As we reported before, PKC activators PMA (16.2 nM) and OAG (50 µM) strongly inhibited the spontaneous GVBD of DOs (PMA, 1.13%, n = 188; OAG, 0.90%, n = 134), whereas these two reagents stimulated GVBD of CEOs (PMA, 69.63%, n = 191; OAG, 81.34%, n = 150) cultured in HX medium (Fig. 1B). The biologically inactive PMA analog 4{alpha}-PDD did not have any effect on meiotic resumption of mouse CEOs as compared with the control (26.89% vs. 28.13%).

PKC activators were reported to induce GVBD in mouse CEOs but not DOs [18]. Whether the MAPK cascade is involved in PKC activator-induced GVBD, as is the case in FSH-induced GVBD [15], has never been studied. As shown in Figures 3 and 4, when mouse CEOs were cultured in HX medium and treated with 16.2 nM PMA for 16 h, MAPK was activated prior to GVBD in both the oocytes and cumulus cells. In the cumulus cells, active MAPK could be detected after 2 h of PMA stimulation and kept active for 12 h (Fig. 3B, lanes 1–5), whereas in the oocytes, no active MAPK and p90rsk could be detected until 8 h of PMA stimulation (Fig. 3A, lanes 1–3). In the cumulus cells, MAPK activity disappeared at 16 h (Fig. 3B, lane 6), whereas in the oocytes active MAPK and p90rsk could be detected after 8 h of FSH treatment and increased as maturation progressed in the following incubation period (Fig. 3B, lanes 3–5). To exclude the possibility that a small number of mature oocytes could account for the positive signal in Western blotting, only the GV-intact oocytes were chosen at 8 h of maturation culture for Western blot. The kinetics of oocyte meiotic maturation based on the same experiments showed that, compared with control groups without PMA treatment, a significant increase in GVBD occurred 8 h after PMA stimulation (Fig. 4) when active MAPK had already been detected in the oocytes and the cumulus cells.



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  		FIG. 3. Effect of PMA treatment on MAPK activation in mouse COCs. A) Oocyte samples collected after treating CEOs with 16.2 nM PMA for 0, 4, 8, 12, and 16 h (lanes 1–5). CEOs were also cultured for 16 h in HX medium containing 16.2 nM PMA + 10 µM U0126 (lane 6) or 16.2 nM PMA + 5 µM calphostin C (lane 7). B) Cumulus cell samples collected after treating COCs with 16.2 nM PMA for 0, 2, 4, 8, 12, and 16 h (lanes 1–6) with 16.2 nM PMA + 10 µM U0126 for 4 and 8 h (lanes 7 and 8), and with 16.2 nM PMA + 5 µM calphostin C for 4 and 8 h (right panels)



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  		FIG. 4. Kinetics of oocyte meiotic resumption after PMA induction. Mouse CEOs were cultured in HX medium for up to 20 h in the presence or absence (control group) of 16.2 nM PMA. GVBD was scored at 4-h intervals

MEK Inhibitor U0126 Inhibited GVBD and MAPK Activation in COCs Induced by FSH and PKC Activators

To investigate the requirement for MAPK activity in PKC activator-induced GVBD, the MEK1/2-specific inhibitor U0126 was added to the HX medium along with PKC activators. As shown in Figure 1, MEK inhibitor U0126 prevented the meiotic resumption induced by FSH (15.46%, n = 97); PMA (7.97%, n = 113); or OAG (16.13%, n = 100). The result with Western blotting showed that the MAPK and p90rsk remained unphosphorylated in CEOs 16 h after culture with FSH or PMA plus U0126 (Fig. 2A, lane 4; Fig. 3A, lane 6). The phosphorylation of MAPK induced by FSH or PMA in cumulus cells was also inhibited at 4 or 8 h after U0126 treatment (Fig. 2B, lanes 6 and 7; Fig. 3B, lanes 7 and 8).

PKC Activity Is Involved in FSH-Induced Meiotic Resumption and MAPK Activation in Mouse COCs

Since both FSH and PKC activators stimulated meiotic resumption and MAPK activation in mouse CEOs, we designed the following experiment to test if the induction of FSH on GVBD and MAPK phosphorylation is mediated by PKC. FSH, PMA, or OAG were added to the HX medium containing PKC-specific inhibitors calphostin C or chelerythrine at the start of maturation culture. The FSH-induced GVBD was decreased by the presence of calphostin C (65.88%, n = 60) or chelerythrine (39.88%, n = 75) in the medium (Fig. 1A). PKC inhibitors calphostin C and chelerythrine also block the inductive effect of PMA and OAG on GVBD of oocytes, as shown in Fig. 1B.

In agreement with its effect on FSH-induced oocyte meiotic resumption, PKC inhibitor calphostin C also inhibited the FSH-induced MAPK activation in both oocytes (Fig. 2A, lane 5) and cumulus cells (Fig. 2B, right panels). PMA-induced MAPK phosphorylation in oocytes (Fig. 3A, lane 7) and cumulus cells (Fig. 3B, right panels) was also blocked by calphostin C treatment. The PKC inhibitor, chelerythrine, which is structurally unrelated to calphostin C, had the same effect on MAPK activity in mouse COCs (data not shown).

FSH and PKC Activators Induce the Cumulus Cells to Secrete a Meiosis-Stimulating Factor by a MAPK-Dependent Pathway

To clarify if cumulus cells induce GVBD by secreting a paracrine factor or exchanging small molecules with oocytes through gap junctions after FSH or PMA stimulation, a coculture system of cumulus cells and DOs was employed. As shown in Figure 5, the GVBD rate in DOs is very low (12.13%, n = 89) at 16 h after coculture of oocytes and cumulus cells in HX medium. The addition of FSH, PMA, or OAG in the medium significantly increased the GVBD rate in DOs cocultured with the cumulus cells FSH (47.67%, n = 86); PMA (60.61%, n = 165); and OAG (61.04%, n = 94). However, if the MEK inhibitor U0126 was also added to the medium with FSH or PKC activators, their stimulatory effect on the meiotic resumption was abolished, and only a small portion of the DOs (FSH treatment, 10.52%, n = 69; PMA treatment, 8.1%, n = 75; and OAG treatment, 9.45%, n = 60) could resume meiosis after coculture with cumulus cells.



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  		FIG. 5. Effect of FSH and PKC activators on GVBD of DOs cocultured with cumulus cells. A group of 30 CEOs isolated from follicles was denuded of their cumulus cells by repeated pipetting. The separated oocytes and cumulus cells were cocultured in 100 µl drops of medium as follows: HX medium, HX medium containing 100 IU/L FSH, HX medium containing 16.2 nM PMA, or HX medium containing 5 µM OAG. In some experiments, 10 µM U0126 was added to the HX medium along with FSH, PMA, or OAG. GVBD was recorded at 16 h of each treatment

In both DOs and cumulus cells cocultured for 16 h in HX medium, no phosphorylated MAPK could be detected (Fig. 6A, lane 1, and Fig. 6B, lane 1). As the case in intact COCs shown in Figures 2B and 3B, FSH and PMA induced the activation of MAPK in cumulus cells at 4 h of culture (Fig. 6B, lanes 2 and 3). However, unlike in separated DOs [11, 15], MAPK and p90rsk were also activated at 16 h of FSH or PMA treatment in DOs cocultured with cumulus cells (Fig. 6A, lanes 2 and 3). Like its effect on GVBD inhibition as mentioned above, MEK inhibitor U0126 also prevented phosphorylation of MAPK in both the cumulus cells and DOs after FSH or PMA treatment in the coculture system (Fig. 6, A and B, lanes 4 and 5).



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  		FIG. 6. Effect of FSH and PMA on MAPK activity in cocultured oocytes and cumulus cells. A group of 30 denuded oocytes and cumulus cells from the same 30 oocytes were cocultured in 100 µl drops of medium. DOs were collected at 16 h after culture, and cumulus cells were collected at 4 h of culture for immunoblotting of MAPK/p90rsk. HX medium, HX medium containing 100 IU/L FSH, and HX medium containing 16.2 nM PMA. In some experiments, 10 µM U0126 was added to the HX medium along with FSH or PMA. Samples represent oocytes (A) and cumulus cells (B) treated as follows: HX medium (lane 1), HX medium with 100 IU/L FSH (lane 2) or 16.2 nM PMA (lane 3), HX medium with FSH plus 10 µM U0126 (lane 4), or 16.2 nM PMA plus 10 µM U0126 (lane 5)

FSH- and PKC-Induced Meiotic Resumption of Oocytes, but not MAPK Activation in Cumulus Cells, Is Dependent on Protein Synthesis

The requirement for protein synthesis in FSH- or PMA-induced GVBD and MAPK phosphorylation in mouse oocytes was assessed by the addition of cycloheximide in culture medium. As shown in Figure 7, isolated fully grown oocytes normally underwent spontaneous GVBD at a high rate (88.45%, n = 186). The spontaneous GVBD of DOs was not influenced by protein synthesis inhibitor CHX (86.87%, n = 99), but was significantly blocked by the cytoplasmic polyadenylation inhibitor cordycepin (1.52%, n = 66). However, CHX strongly inhibited both the FSH-induced GVBD (1.06%, n = 94) and the PMA-induced meiotic resumption (3.09%, n = 97) of CEOs. The cytoplasmic polyadenylation inhibitor cordycepin could also significantly inhibit the PMA-induced GVBD of CEOs (1.23%, n = 81), but did not have any statistical significant effect on CEOs cultured with FSH (81.61%, n = 87). Both CHX and cordycepin prevented the MAPK/p90rsk activation in DOs cultured in HX-free medium (Fig. 8, lanes 5 and 6), although the spontaneous GVBD was not inhibited by CHX. CHX and cordycepin were efficient in inhibiting the MAPK/p90rsk activation induced by FSH or PMA in CEOs (Fig. 8, left panels, lanes 1–4), but in cumulus cells, the activation of MAPK induced by PMA and FSH at 4 h of culture was not inhibited by CHX or cordycepin (Fig. 8, right panels).



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  		FIG. 7. Effect of CHX on meiotic resumption of mouse oocytes. Mouse CEOs were cultured in HX medium supplemented with 100 IU/L FSH, FSH + 5 µg/ml cycloheximide (CHX), FSH + 50 µM cordycepin (dA), 16.2 nM PMA, PMA + 5 µg/ml CHX, or PMA + 50 µM dA. Some DOs were cultured in HX-free medium supplemented with 5 µg/ml CHX or 50 µM dA. GVBD was observed at 16 h of culture



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  		FIG. 8. Effect of CHX and cordycepin (also named deoxyadenosine [dA]) on MAPK activation in mouse COCs. CEOs were collected for immunoblotting after the following treatment for 16 h: 16.2 nM PMA + 50 µM dA, PMA + 10 µg/ml CHX, 100 IU/ml FSH + dA, or FSH + CHX (left panels, lanes 1–4). Cumulus cells after the same treatment for 4 h were also collected for Western blotting (right panels). Lanes 5 and 6 of left panels represent the samples of DOs treated with 50 µM dA or 10 µg/ml CHX for 16 h. A sample of MII-arrested mouse oocytes was loaded as the positive control (lane 7 of left panels)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In mammals, the role of protein kinase C and MAPK cascade in oocyte meiotic resumption has not been clarified in detail. A number of our previous studies have focused on the function of intracellular messengers using systems in which oocytes were induced to mature spontaneously [11, 12, 19]. Spontaneous maturation involves removal of the inhibitory influence imposed by the follicular environment. However, this model does not reproduce accurately the normal mechanisms operating in vivo, and therefore it does not appear to be the most appropriate approach. In contrast, systems in which meiotic maturation is induced by gonadotrophins or other agents under conditions that prevent spontaneous maturation offer a more physiological model. In such a model, reinitiation of meiosis, as it presumably occurs in vivo, is activated via the interaction between the oocyte and cumulus cells. Previous reports indicated that MAPK activity in cumulus cells is necessary for the FSH-induced meiotic resumption of CEOs, but not the spontaneous GVBD of denuded oocytes [15, 17, 39]. However, the regulation and functional roles of MAPK in cumulus cells is so far unknown. Furthermore, it was recognized that PKC activators inhibit spontaneous GVBD in DOs but stimulate the meiotic resumption in CEOs through mediation of cumulus cells [18], but the signal transduction process of PKC in cumulus cells that leads to oocyte meiotic resumption is unknown. Here we provide evidence that PKC activators, PMA and OAG, could activate MAPK in cumulus cells in the absence of FSH stimulation, and MEK inhibitor U0126 efficiently prevented the PMA- or OAG-induced GVBD of CEOs. PKC inhibitors, calphostin C and chelerythrine, blocked the FSH-induced meiotic resumption and MAPK activation induced by either FSH or PKC activators. Based on these findings, we hypothesize that PKC activity in cumulus cells might lead to the meiotic resumption of oocytes through mediation of MAPK, and in gonadotropin-induced meiotic resumption, PKC is involved in the regulation of FSH-induced MAPK activation. In other words, PKC might be the linkage between FSH stimulation and MAPK activation in induced meiotic resumption of mouse CEOs. This hypothesis is supported by previous reports. Su et al. [3] suggested that PKC and intracellular calcium (which is a well-known PKC activator) are involved in FSH-mediated GVBD of pig CEOs cultured in HX-supplemented medium. Similarly, inhibition of phosphoinositide metabolism or chelation of intracellular calcium could also block FSH-induced meiotic resumption of mouse CEOs [40].

MAPK is an important component of many signal transduction pathways that transmit various stimuli and regulate cell cycle progression and cell proliferation in eukaryotic cells, but the function of this kinase in mediating the FSH- or PKC-induced meiotic resumption of oocytes is unclear. In this report, when denuded oocytes were cocultured with cumulus cells in HX-supplemented medium containing FSH or PKC activators, GVBD could be induced, and addition of MEK inhibitor U0126 to the coculture system abolished this inductive effect. MAPK phosphorylation was detected in the cumulus cells after FSH or PMA stimulation, whereas U0126 reversed this effect. These results suggest that FSH or PKC may stimulate the cumulus cells to secrete a paracrine factor that induces meiotic resumption of oocytes, and even more important, the secretion of this maturation-inducing factor in cumulus cells is triggered by MAPK. Other groups also reported that cumulus cells secreted a meiosis-activating substance (MAS) when stimulated by FSH, EGF, or cAMP analogues [6, 41]. In further investigations, follicular fluid MAS (FF-MAS) was identified within the preovulatory follicles [42]. It is probably produced by cumulus cells and could induce in vitro resumption of meiosis in denuded and cumulus-enclosed mouse oocytes inhibited by hypoxanthine, 3-isobutyl-1-methylxanthine, or dibutyric cyclic AMP. Here we report that PKC activators may stimulate the cumulus cells to secrete the positive factor in the same manner as FSH does, and MAPK activity might be involved in the signal transduction pathway that leads to the synthesis and secretion of this positive factor stimulated by FSH. The cooperation of PKC and MAPK in response to the extracellular stimulation has been reported in many cell types [43, 44], including cultured human follicular cells [45].

In germ cells, Mos serves as the MAPK leading to the activation of MAPK cascade [46], and p90rsk is the best known physiological target of MAPK in Xenopus oocytes [47, 48]. MAPK cascade, by regulating the activity of other enzymes mainly at the posttranslational level, is essential for the meiotic cell cycle control of vertebrate oocytes. But in somatic cell lines, MAPK may play a role in the regulation of cell proliferation or differentiation in various ways, including the regulation of gene transcription by phosphorylating transcription factors such as cAMP-response element binding protein and proto-oncogene products c-Jun and c-Fos [49]. For example, EGF induces c-fos and c-jun mRNA via a Raf-1/MEK1/ERK-dependent pathway in bovine luteal cells [50]. Therefore, due to their somatic origin, cumulus cells may be different from oocytes in regard to MAPK functions and pathways. In our experiments, phosphorylation of p90rsk following MAPK activation was only detected in oocytes, but not in the cumulus cells, although this kinase existed in both cell types. Thus, in cumulus cells MAPK might phosphorylate target molecules other than p90rsk in promoting meiotic initiation of oocytes. 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 is exactly the case in our experiments. Eukaryotic protein synthesis inhibitor cycloheximide prevented the GVBD of CEOs induced by either FSH or PMA, although the MAPK activation in cumulus cells was not affected. This result inferred that activated MAPK stimulated the production of new proteins in cumulus cells, which may be essential for synthesis and/or secretion of positive factor(s) for meiosis induction, and then the meiotic reinitiation of oocytes.

Maternal mRNA translation is regulated largely by cytoplasmic polyadenylation. Accumulation of cyclin A and B requires cytoplasmic polyadenylation, a process that can be inhibited by cordycepin (also named deoxyadenosine [dA]) [51]. Cordycepin is metabolized to 3'-dATP in the cell and incorporated into the poly(A) tail during polyadenylation. Once 3'-dA is added to the poly(A) terminal, further addition of adenosine is inhibited [51]. We found that spontaneous GVBD, but not the FSH-induced meiotic resumption, was sensitive to cordycepin, which is in accordance with the former reports in mouse oocytes [8]. In contrast, PMA-induced GVBD was significantly inhibited by cordycepin, indicating that the cytoplasmic polyadenylation of mRNAs is essential for the PMA-induced meiotic resumption of CEOs. These data also suggested that FSH-induced and PMA-induced meiotic resumption in mouse COCs are differently mediated in some steps. The different effects of CHX and cordycepin on FSH-induced and spontaneous GVBD of mouse oocytes were also reported by other laboratories with the same results as us, but there is still no explanation about these phenomena. As an analog of ATP, cordycepin may influence intracellular events other then mRNA polyadenylation and prevent GVBD in DOs. In agreement with a previous report, the spontaneous GVBD of DOs was not inhibited by CHX, but we noticed that although DOs could undergo GVBD in HX-free medium supplemented with CHX, MAPK failed to be activated even after 16 h of culture. These results revealed that synthesis of new proteins is the prerequisite for MAPK activation in oocytes, but that MAPK activation in cumulus cells is independent of mRNA polyadenylation and protein synthesis. In the present experiments we also showed that MAPK was promptly activated in the cumulus cells much earlier than in the oocytes. Its activation was detected at 2 h of PMA stimulation, and maximum activation was detected between 4 and 8 h. How PKC stimulates MAPK in cumulus cells is still unknown. The protein p21ras is an upstream activator of MAPK in Xenopus oocytes [52, 53], in which it can induce meiotic maturation [54]. The P21ras inhibitor lovastatin inhibits the FF-MAS-induced meiotic maturation, but not the spontaneous maturation of mouse oocytes [8]. Phospholipase C{gamma} (PLC{gamma}) inhibitor U73122 also inhibits the FSH-induced GVBD in mouse CEOs [8]. Whether PKC induce MAPK activation by activating G-protein or PLC family members is still waiting to be tested. Furthermore, unlike in oocytes, MAPK activity within the cumulus cells decreased quickly after 12 h of PMA stimulation. Similar dynamics of MAPK phosphorylation/dephosphorylation were also reported in cumulus cells after FSH stimulation [15]. At present we still cannot explain the role of MAPK inactivation within the cumulus cells during FSH or PMA-induced mouse oocyte meiotic resumption. It may be the result of negative feedback in cumulus cells after long-term exposure to extracellular stimulation. The negative feedback mechanism works widely in cells to maintain homeostasis of organisms. The FSH- or PMA-induced oocyte meiotic resumption is different from spontaneous oocyte maturation in that MAPK and p90rsk are activated before GVBD, which is an event that normally occurs 2 h after GVBD in spontaneous maturing mouse oocytes [28, 55]. Although MAPK was activated before FSH- or PMA-induced GVBD in CEOs, it is not likely that the MAPK activity in oocytes is absolutely essential for meiotic resumption because GVBD could occur normally in oocytes from c-mos knockout mice. Whether the mechanisms responsible for MAPK activation during meiotic resumption are different between spontaneous and induced maturation of oocytes needs further clarification.

Some specific concerns should also be addressed in this study. First, as shown in Figures 1 and 2, PKC inhibitors abolished MAPK phosphorylation but could not reduce GVBD to the control level as MEK inhibitor U0126 did. One possibility is that PKC inhibition only accounts for a portion of a larger unexplored mechanism, and other signaling pathways may also be involved in the FSH-induced meiotic resumption of oocytes. Second, although GVBD could be induced when denuded oocytes were cocultured with cumulus cells in the presence of FSH or PMA, we cannot rule out the possibility that treatment of cumulus cells with FSH or PKC activators stimulates cellular metabolism and leads to increased removal of an inhibitory factor in the medium. Third, all of the experiments herein have been carried out using hypoxanthine to maintain meiotic arrest. Although this is a physiological inhibitor, it is important to determine in future studies if there is a similar meiotic response to FSH or PKC activators when arrest is maintained with other inhibitors, such as isobutylmethylxanthine, forskolin, or dibutyric cyclic AMP. Finally, we still cannot conclude from the results whether the release of a paracrine factor by cumulus cells is the primary mechanism for meiotic induction induced by FSH or PKC activator stimulation, or whether it only contributes to the effect along with other mechanisms. Other investigators have proposed that a positive meiosis-inducing stimulus produced in the cumulus cells is transferred to the oocyte through gap junctions [7, 56–58]. Further rigorous tests are necessary before a definite mechanism is clarified.

Taken together, the present study reveals a possible signaling pathway that mediates FSH-induced meiotic resumption of mouse oocytes: after binding with its membrane integrated receptor in cumulus cells, FSH leads to the activation of MAPK with the mediation of PKC. Activation of MAPK induces the synthesis of certain proteins followed by the secretion of a meiosis-stimulating factor by cumulus cells. The undefined soluble factor induces the oocytes to resume meiosis. The possible signal transduction pathway that leads to oocyte meiotic resumption is illustrated in Figure 9.



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  		FIG. 9. Hypothesis of signal transduction leading to meiotic resumption in mouse COCs stimulated by FSH. FSH leads to the activation of PKC in cumulus cells by an undefined mechanism, and then activation of PKC stimulates the phosphorylation of MAPK, which induces the biosynthesis of certain proteins. Following these changes in the cumulus cells, a positive factor is secreted by cumulus cells and contributes to meiotic induction in a paracrine fashion. Activity of MAPK appears before GVBD in oocytes in FSH- or PMA-induced meiotic maturation, but this kinase activity in oocytes is not absolutely necessary for the initiation of GVBD


   ACKNOWLEDGMENTS
 
The authors thank Hui Zou and Chao Tong from the University of Texas Southwestern Medical Center for their enlightening discussions and kindly help in the preparation of the manuscript.


   FOOTNOTES
 
1 Supported by the Special Funds for Major State Basic Research Project (973) of China (G1999055902), National Natural Science Foundation of China (30225010), and Knowledge Innovation Project of the Chinese Academy of Sciences (KSCX2-SW-303 and KSCX-IOZ-07). Back

2 Correspondence: Qing-Yuan Sun, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. FAX: 8610-6256-5689; sunqy1{at}yahoo.com Back

Received: 30 October 2003.

First decision: 29 November 2003.

Accepted: 10 December 2003.


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J. Endocrinol., May 1, 2008; 197(2): 409 - 419.
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S. Panigone, M. Hsieh, M. Fu, L. Persani, and M. Cont