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Biology of Reproduction 65, 358-365 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Involvement of MEK-Mitogen-Activated Protein Kinase Pathway in Follicle-Stimulating Hormone-Induced but Not Spontaneous Meiotic Resumption of Mouse Oocytes1

You-Qiang Sua, Sara Rubinsteina, Ayala Luriaa, Yehudit Laxa, and Haim Breitbarta

a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

ABSTRACT

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.

developmental biology, FSH, kinases, meiosis, oocyte development, ovum, signal transduction

INTRODUCTION

Mammalian oocytes are arrested at the diplotene stage of the first meiotic prophase (late G2 phase) around the time of birth and remain in this late G2 phase of the first meiotic cell cycle until just prior to ovulation, when meiosis reinitiation (G2-M phase transition) is triggered by the preovulatory gonadotropin surge [1, 2]. However, the molecular mechanism(s) underlying this phenomenon are still not very clear. In vitro, fully grown meiotic competent oocytes can undergo spontaneous maturation, when liberated from Graffian follicles and cultured in a suitable medium [3, 4]. Oocytes can also resume meiosis under the stimulation of gonadotropins when oocyte spontaneous maturation is prevented by a number of inhibitory agents, such as cAMP analogues, phosphodiesterase (PDE) inhibitors and purines [5], or by oocyte coculture with follicular somatic components [6, 7]. This process requires the presence of cumulus cells that are the source of a positive meiosis-inducing signal(s) [8].

Oocyte meiotic cell cycle progression is thought to be orchestrated by protein phosphorylation-dephosphorylation cascades [9]. MPF (maturation- or metaphase-promoting factor) and MAP (microtubule-associated or mitogen-activated protein) kinase are two key molecules involved in this process and have recently been the subject of numerous studies. Based on the data from the reports in all species studied to date, it is clear that MPF plays a pivotal role in driving the oocyte meiotic cell cycle progression because it is detected in the oocyte and its kinetics of activation precede or coincide with germinal vesicle breakdown (GVBD) [10]. However, MAP kinase has also been reported to be activated during oocyte maturation and suggested to play a crucial role in controlling oocyte meiotic cell cycle progression [10]. In Xenopus oocytes, MAP kinase activation is essential for progesterone-induced oocyte meiotic resumption [11]. However, in mammalian oocytes, reports on the role of MAP kinase during oocyte meiotic resumption are not definitive. Indeed, depending on the animal species and culture systems used, the evidence is conflicting. During oocyte spontaneous maturation in the mouse, rat, and goat, MAP kinase is activated after GVBD and MPF activation [12–17], suggesting that MAP kinase may not be directly involved in the resumption of meiosis but rather in the events post-GVBD. However, during equine, porcine, and bovine oocyte spontaneous maturation, MAP kinase is activated before GVBD [18–20]. In some special circumstances, MAP kinase has also been reported to be activated before GVBD, for instance, in okadaic acid-induced maturation of mouse meiotic-competent or incompetent oocytes, and MEK RNA or MOS protein injection-induced mouse oocyte maturation when meiosis is prevented by a PDE inhibitor [21–23]. The role of MAP kinase in the events of oocyte meiotic resumption is therefore unclear.

However, most of the information in the above reports is achieved by means of a spontaneous oocyte maturation paradigm that differs physiologically from gonadotropin-induced oocyte meiotic resumption. The former is thought to be a relatively passive response to the artificial removal of a meiotic suppressor, whereas the latter is more active, presumably requiring gonadotropin-triggered production of a meiosis-inducing stimulus [24–27]. It has been demonstrated in vitro that FSH can stimulate cumulus cells of mouse cumulus-oocyte-complexes (COC) to generate a meiosis-inducing signal(s) that acts on the oocyte and induces it to override the arrest of hypoxanthine, a natural meiotic inhibitory substance existing in follicular fluids, and resume meiosis [8, 28]. Several lines of evidence suggest that spontaneous and FSH-induced oocyte meiotic resumption are two quite different events. For instance, in a series of reports detailing the influence of energy substrate on oocyte maturation, it has been established that FSH-induced, but not spontaneous, maturation requires the presence of glucose in the mouse COC [29–31]. FSH-induced, but not spontaneous, meiotic resumption in the mouse oocyte requires the activation of the phosphoinositide pathway and mobilization of intracellular calcium [32]. The discrepancies demonstrated in the above reports suggest that biochemical pathways required for gonadotropin-induced oocyte meiotic resumption may not be required for spontaneous meiotic resumption, and oocyte spontaneous maturation might be a deficient system for studying the regulation of oocyte meiotic cell cycle. The question of whether MAP kinase is involved in FSH-induced oocyte meiotic resumption remains unresolved.

Hence, the present study was undertaken to investigate the possible role of MAP kinase during FSH-induced oocyte meiotic resumption and compare it with that of spontaneous oocyte meiotic resumption. The results demonstrated in this study indicate that the MEK-MAP kinase pathway is involved in FSH-induced, but not spontaneous, oocyte meiotic resumption in the mouse.

MATERIALS AND METHODS

Chemicals

Minimum essential medium eagle (MEM), eCG, L-glutamine-penicillin-streptomycin solution, sodium pyruvate, BSA, hypoxanthine, and FSH (from human pituitary) were purchased from Sigma Chemical Co. (St. Louis, MO). PD 98059 and U0126 were purchased from Calbiochem Chemical Corporation (La Jolla, CA). Stock solution of FSH (100 IU/ml) was prepared in MEM and stored at -20°C. Stock solution of PD 98059 and U0126 (20 mM and 2 mM, respectively) were prepared in anhydrous dimethylsulfoxide (DMSO) and stored desiccated at -20°C. Prior to use, they were diluted with MEM, and the final concentration of DMSO was less than 0.2%. Monoclonal anti-MAP kinase activated (diphosphorylated ERK1 and 2) antibody, lot number 069HR007, was purchased from Sigma Israel Chemical Ltd. (Rehovot, Israel). Polyclonal anti-ERK2 antibody, lot number 1276, was purchased from Santa Cruz Biotechnology Incorporation (Santa Cruz, CA). Donkey anti-rabbit and goat anti-mouse IgG were purchased from Jackson Immune Research Laboratories, Inc. (Bar Harbor, ME) Super Signal chemiluminescence substrate was purchased from the Pierce Company (Chicago, IL).

Oocyte Isolation and Culture

Immature female BALB/c mice (22–24 days old), were used for all experiments. All procedures involving mice were conducted in accordence with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy of Sciences, Bethesda, MD). Follicle development was stimulated by i.p. injection with 5 IU eCG. Mice were killed 44–48 h later by cervical dislocation, and their ovaries were dissected and transferred to pre-equilibrated culture medium. Oocytes were released by puncturing large antral follicles with sterile 30-gauge needles. COC were collected, and denuded oocytes (DO) were obtained by repeated pipetting COC through a fine-bore pipette. COC and DO were washed through three additional changes of medium and then transferred in a small volume to four-well plastic culture dishes (Nunclon, Roskilde, Denmark) containing 500 µl of the appropriate culture medium. Medium used in all experiments was MEM containing 25 mM Hepes, 0.3% BSA, 0.23 mM pyruvate, 2 mM glutamine, and 100 IU/L penicillin and streptomycin. Oocytes were cultured at 37°C, in an atmosphere of 5% CO2 and 100% humidity.

Experimental Treatments of the Oocytes

To examine the possible role of MAP kinase in FSH-induced oocyte maturation, COC were cultured in 4 mM hypoxanthine (HX)-supplemented medium (HX-medium) to suppress the oocytes undergoing spontaneous meiotic resumption, and oocyte meiotic resumption was induced by exposing COC to 100 IU/L FSH. MAP kinase activation was inhibited by adding different doses of MAPKK (MEK) inhibitor, U0126 or PD98059, into the culture medium at the begining of culture. To investigate the role of MAP kinase in spontaneous oocyte meiotic resumption, COC or DO were cultured in the medium without HX, and MAP kinase activation was inhibited by adding U0126 into the medium at the beginning of culture. Culture time for both experiments was 20 h. At the end of culture, the incidence of oocyte GVBD and first polar body (PB1) extrusion was scored under an inverted microscope after the oocytes were denuded of their cumulus cells. Cumulus cells were separated by centrifuging at 14 000 rpm after DO removal. Samples of oocytes and cumulus cells for Western blotting were collected and stored at -70°C until use.

To study the kinetics of MAP kinase activation within oocytes and cumulus cells in the process of FSH-induced oocyte maturation, COC were cultured in HX-medium in the presence of 100 IU/L FSH for up to 24 h. During the culture, the incidence of oocyte GVBD and PB1 extrusion was scored at different culture time points, and samples of oocytes and cumulus cells were collected at the same time points and subjected to western blotting for detecting the activity of MAP kinase with the anti-active MAP kinase antibody.

Electrophoresis and Western Blot Analysis

Proteins from 30 DO or from cumulus cells of 30 COC were extracted with double-strength electrophoresis sample buffer composed of 125 mM Tris, pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol, 10% (v/v) ß-mercaptoethanol, 0.004% (w/v) bromophenol blue, and the lysates were heated to 100°C for 4 min. After cooling down on ice for 4 min and centrifuging at 14 000 rpm for 5 min, samples were frozen at -70°C until use. The proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel for 50 min at 179 V and electrophoretically transferred onto a piece of Protran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) for 35 min at 20 V using a Semi-Dry Trans-Blot apparatus (Bio-Rad Laboratories, Hercules, CA). After blocking for 1 h with 5% nonfat dry milk in 20 mM Tris, 137 mM NaCl (TBS, pH 7.6), the membrane was incubated overnight at 4°C with monoclonal anti-MAP kinase, activated (diphosphorylated ERK1 and 2) antibody diluted 1:1000 with TBS containing 0.5% BSA. This antibody reacts specifically with the active, double-phosphorylated form of the MAP kinases (ERK1 and 2). After three washes of 10 min each in TBS containing 0.1% Tween-20 (TBST), the membrane was incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG diluted 1:2000 in TBST. The membrane was then washed three times in TBST and visualized using the enhanced chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL).

For reprobing MAP kinase, the blots were stripped of the bound antibodies by washing in a stripping buffer (100 mM ß-mercaptoethanol, 20% SDS, 62.5 mM Tris, pH 6.7) and then reprobed with polyclonal anti-ERK2 antibody diluted 1:3000, using the same procedure as described above. The second antibody used for reprobing was HRP-conjugated donkey anti-rabbit Ig G diluted 1:5000 in TBST.

Oocyte Assessment and Statistical Analysis

Oocytes were assessed for maturation at the end of the culture period by removing cumulus cells and scoring them for GVBD (the resumption of nuclear maturation) or PB1 extrusion (indicating progression to metaphase II). Each oocyte maturation experiment was conducted at least three times with 30 oocytes per group per experiment, and the data were reported as the mean percentage of GVBD or PB ± SEM. All frequencies were subjected to arcsin transformation, and the differences between treatment and control groups were statistically analyzed by one-way pairwise (Student t-test) comparison. A P value less than 0.05 was considered to be significant.

RESULTS

Inhibition of MAP Kinase Activation Blocks FSH-Induced Oocyte Meiotic Maturation

As shown in Figure 1, when mouse cumulus cell-enclosed oocytes (CEO) were cultured in HX-medium for 20 h, less than 30% of the oocytes underwent GVBD and less than 25% of the oocytes extruded PB1. Under the stimulation of 100 IU/L FSH, the percentages of the oocyte underwent GVBD and PB1 extrusion increased to more than 79% and 40%, respectively. These results are consistent with previous reports [8, 28]. When mouse CEO were treated with PD98059 or U0126, two specific MAPKK inhibitors, FSH-induced oocyte meiotic maturation was dose-dependently inhibited by either of the inhibitors. However, no significant inhibitory effects could be observed when the oocytes were cultured in HX-medium in the absence of FSH (Fig. 1). PD98059 (Fig. 1A) at a concentration of 2–200 µM resulted in a statistically significant inhibition of both GVBD and PB1 extrusion (GVBD: 51.8–60.5% versus 79.1%; PB1: 10.6–27.2% versus 40.0%, P < 0.05). U0126 (Fig. 1B), a new generation of specific MAPKK inhibitor, exerted a more potent inhibitory effect on FSH-induced oocyte meiotic resumption. At a concentration of 1 µM or above, it could dramatically inhibit FSH-induced GVBD and PB1 extrusion (GVBD: 24.5–50.5% versus 87.5%; PB1: 16.9–20.1% versus 54.2%, P < 0.05).



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  		FIG. 1. Effects of MAPKK (MEK) inhibitor, PD98059 or U0126, on mouse oocyte meiotic resumption. Mouse CEO were treated with increasing concentrations of PD98059 (A) or U0126 (B) and cultured for 20 h in HX-medium supplemented with (circles) or without (squares) 100 IU/L FSH. Data are shown as mean percentage of GVBD (or PB1, as shown in the inserts) ± SEM of five independent experiments. *P < 0.05, compared with control group (0 µM PD98059- or U0126-treated group). No significant differences were detected between control groups and any MEK inhibitor-treated groups in HX-medium without FSH addition

In order to test whether MAP kinase within the oocyte and cumulus cell is activated during FSH-induced mouse oocyte meiotic maturation and whether the inhibitor of MAPKK (MEK) can inhibit this activation, the activity of MAP kinase was detected by Western blot analysis using the specific anti-active MAP kinase antibody. The results (Fig. 2, upper panel) showed that in the presence of 4 mM HX, without FSH addition, low MAP kinase activity was detected within the oocytes at the end of 20 h of culture. FSH (100 IU/L) dramatically activated MAP kinase in the oocytes. When COC were treated with FSH plus U0126, FSH-stimulated MAP kinase activation within the oocytes was inhibited by U0126 in a dose-dependent manner. MAP kinase activity within the oocytes was almost completely abolished by 5 µM U0126. However, at the end of 20 h culture, no active MAP kinase was detected in the cumulus cells with FSH treatment. The change in active MAP kinase was not due to the change in the amount of this enzyme because no differences were observed when the same samples were reprobed with anti-ERK antibody (Fig. 2, lower panel).



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  		FIG. 2. Effect of MAPKK inhibitor, U0126, on MAP kinase activation within the oocytes and cumulus cells during FSH-induced oocyte meiotic resumption. Mouse COC were cultured in HX-medium for 20 h in the presence of 100 IU/L FSH and increasing concentrations of U0126. 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 COC were loaded into each lane. The experiment was performed three times with similar results. Blots were incubated with a monoclonal anti-active MAP kinase antibody to show MAP kinase activity (upper panel). The same blots were subsequently stripped and reprobed with polyclonal anti-ERK antibody (lower panel). The position of ERKs is indicated on the left. The same treatments were applied in all subsequent Western blot figures. F0: control group without FSH or U0126 treatment; F100 + U0, U1, U2.5, U5, U10, U20: treatments with 100 IU/L FSH plus 0, 1, 2.5, 5, 10, or 20 µM U0126.

Inhibition of MEK-MAP Kinase Pathway Does Not Block Spontaneous Meiotic Resumption of Mouse Oocytes

When CEO or DO were cultured in HX-free medium for 20 h, almost all the oocytes underwent GVBD spontaneously (94.3% of CEO or 98.2% of DO, respectively, see Fig. 3, A and B). MAP kinase activity within the oocytes could be detected by Western blot analysis using the specific anti-active MAP kinase antibody (Fig. 4, upper panel). U0126, at a concentration of 5–20 µM that could effectively block FSH-induced oocyte meiotic resumption did not inhibit spontaneous meiotic resumption of either CEO (Fig. 3A) or DO (Fig. 3B) after 20 h of culture, despite the fact that the activity of MAP kinase in the oocytes was totally inhibited (Fig. 4, upper panel). No inhibitory effect of U0126 on spontaneous GVBD was observed in the earlier culture period before 20 h (data not shown).



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  		FIG. 3. Effect of MAPKK inhibitor, U0126, on spontaneous oocyte meiotic resumption. Mouse DO (A) or CEO (B) were cultured in medium without HX for 20 h in the presence of U0126 (0, 5, 20 µM). Data are shown as the mean percentage of GVBD ± SEM of three experiments. There were no significant differences between 0 µM U0126 treatment and five, 20 µM U0126 treatments



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  		FIG. 4. Effect of treatment with U0126 on MAP kinase activation in oocytes during spontaneous meiotic resumption. Mouse DO (A) or CEO (B) were cultured in medium without HX for 20 h in the presence of U0126 (0, 5, 20 µM). Samples for immunoblotting were collected at the end of culture after the incidence of GVBD was evaluated. This figure represents one of the similiar results in three independent experiments. U0, U5, U20: 0, 5, 20 µM U0126 treatments

Kinetics of MAP Kinase Activation in the Oocytes and Cumulus Cells During FSH-Induced Oocyte Meiotic Resumption

As shown in Figures 5 and 6, when mouse CEO were cultured in HX-medium and treated with 100 IU/L FSH for 24 h, MAP kinase in both the oocytes and cumulus cells was activated prior to GVBD. In the cumulus cells, active MAP kinase could be detected after 1 h of FSH stimulation and kept active until 8 h; while in the oocytes, no active MAP kinase could be detected until 8 h of FSH stimulation (Fig. 5, A and B, upper panels). In the cumulus cells, MAP kinase activity began to decrease at 12 h and disappeared after 16 h; while in the oocytes it could be detected at 8 h of FSH treatment and increased as maturation progressed in the following incubation period (Fig. 5, A and B, upper panels). In the control groups without FSH treatment, no (or little, if any) active MAP kinase could be detected in either the oocytes or cumulus cells during the 24-h culture (Fig. 5, C and D, upper panels). The kinetics of oocyte meiotic maturation based on the same experiments showed that compared to control groups without FSH treatment, a significant increase in GVBD and PB1 extrusion began at 12 h of FSH stimulation (Fig. 6), which was 4 h later than MAP kinase activation in the oocyte and even much later than in the cumulus cells.



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  		FIG. 5. Kinetics of MAP kinase activation within the oocytes and cumulus cells during oocyte in vitro maturation. Mouse COC were cultured in HX-medium for only 3 h in the presence (A) or absence (C) of 100 IU/L FSH. Samples of oocytes and cumulus cells for immunoblotting were collected at 0, 0.5, 1, 2, and 3 h during culture after evaluation of maturation status. Or COC were cultured in HX-medium for up to 24 h in the presence (B) or absence (D) of 100 IU/L FSH. Samples of oocytes and cumulus cells for immunoblotting were collected every 4 h (0, 4, 8, 12, 16, 20, 24 h) after evaluation of oocyte maturation status. The experiment was performed three times with similar results



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  		FIG. 6. Kinetics of oocyte in vitro maturation. Mouse COC were cultured in HX-medium for up to 24 h in the presence of 100 IU/L FSH (FSH treatment) or without FSH (control group). GVBD (A) or PB1 extrusion (B) was scored at 4-h intervals. Data are represented as mean percentage of GVBD (PB1) ± SEM of three independent experiments. *P < 0.05, compared with each corresponding group in the control

DISCUSSION

In most mammalian species, oocytes contain two isoforms of MAP kinases, ERK1 and ERK2, that have been reported to be activated during oocyte meiotic maturation and implicated to be functional for the regulation of oocyte meiotic cell cycle progression. However, reports on the role of MAP kinase during oocyte meiotic resumption are not definitive, depending, for example, on the variety of animal species. Furthermore, most information from previous reports was achieved using a spontaneous oocyte maturation model in vitro. The role of MAP kinase in gonadotropin-induced oocyte meiotic resumption has not been identified. In the present study, using the MAPKK (MEK) inhibitor, U0126, and a recently developed very sensitive active-MAP kinase Western blot assay, we have investigated the role of MAP kinase in FSH-induced mouse oocytes in in vitro meiotic resumption and compared with that with spontaneous oocyte meiotic resumption. Our studies provide the first demonstration that inhibition of MAP kinase activation blocks FSH-induced, but not spontaneous, oocyte meiotic resumption. Under the conditions of FSH-induced oocyte meiotic resumption, activation of MAP kinase within both the oocytes and the cumulus cells occurs before the appearance of GVBD, and MAP kinase in the cumulus cells is activated even much earlier than in the oocytes. These results suggest that the MEK-MAP kinase pathway is involved in gonadotropin-induced oocyte meiotic resumption, while it is not required for spontaneous meiotic resumption.

MAP kinase is an important component of many signal transduction pathways that transmit various extracellular signals to their intracellular targets and regulate cell cycle progression and cell proliferation in eukaryotic cells. In Xenopus oocytes, the activation of MAP kinase is essential for progesterone-induced MPF activation and oocyte meiotic resumption [11]. Yet, Fisher et al. [33] reported that suppressing MAP kinase activation by treatment with geldanamycin or by overexpression of phosphatase Pyst1 cannot inhibit progesterone-induced Xenopus oocyte maturation. However, because it is not clear whether these treatments can directly activate Cdc2 kinase, which induce the resumption of meiosis, interpretation of these data should be with caution. In equine, porcine, and bovine oocytes, MAP kinase and MPF are also activated concurrently before GVBD and may both be required for oocyte meiotic resumption [18–20]. However, in the mouse, previous reports showed that MAP kinase is activated after MPF activation and GVBD, suggesting that MAP kinase activation may not be necessary for oocyte meiotic resumption [12–14, 17]. However, it should be pointed out that all previous reports regarding MAP kinase activation in mouse oocytes use the spontaneous maturation paradigm, where GVBD occurs without hormonal stimulation. While in the abovementioned reports regarding MAP kinase activation in equine, porcine, and bovine oocytes, gonadotropins were also used in the culture medium. In mammals, oocyte spontaneous meiotic resumption and gonadotropin-induced meiotic resumption are two quite different events. The former involves the artificial removal of the inhibitory influence imposed by follicular environment, without considering the interaction between the oocyte and its follicular components in vivo [27]. In contrast, the latter necessitates a positive signal that overrides the inhibitory environment of the follicle [25, 26]. In the present report, we have investigated the role of MAP kinase during gonadotropin-induced oocyte meiotic resumption by culturing the oocytes in HX-supplemented medium, where meiotic resumption was induced by FSH treatment. This model, initially reported by Eppig and Downs [34], is now widely used for studying the mechanism of oocyte meiotic maturation. We found that inhibition of MAP kinase activation could dramatically inhibit FSH-induced oocyte meiotic resumption, suggesting that the MEK-MAP kinase pathway is involved in this process. This hypothesis was further confirmed by evidence that the activation of MAP kinase in both the oocytes and cumulus cells was detected earlier than GVBD. However, inhibition of MAP kinase activation could not block spontaneous meiotic resumption. These results and recent reports that show that FSH-induced oocyte meiotic resumption is quite different from the spontaneous meiotic resumption in both signal transduction and requirement of energy substrates [29–32] imply that spontaneous maturation may not be an appropriate system for studying the regulation of oocyte meiotic cycle. Spontaneous meiotic resumption might be induced by removal of the oocytes from the inhibition of cAMP leading to the activation of MPF because cAMP can keep MPF in an inactive form and inhibit GVBD [35, 36]. MAP kinase activation during oocyte spontaneous meiotic maturation may be induced by MPF through a kind of positive feedback mechanism as reported in Xenopus oocytes [11].

MAP kinase activation is the result of a complex cascade of kinase activation that includes, immediately upstream, MAPKK (MEK), which in turn is phosphorylated by one of the MAPKKK. In Xenopus oocytes, at least three pathways can lead to MAP kinase activation. One pathway involves MOS and is activated mainly after progesterone stimulation [37]. The other two pathways depend on a receptor tyrosine kinase, of which one is RAF dependent and the other one is RAF independent [38–40]. All three pathways induce MAP kinase activation via MAPKK (MEK) in vivo and in vitro [37, 40–43]. Thus, the proto-oncogene products, MOS and RAF, are two candidates for MAPKKK in Xenopus oocytes. Both MOS and RAF have been reported to be activated in progesterone-treated Xenopus oocytes and are essential for progesterone-induced MAP kinase activation and oocyte maturation [41, 44]. In contrast to Xenopus oocytes, in Mos-/- mice, oocytes can undergo GVBD normally [45, 46]. No or low MAP kinase activity can be detected in Mos-/- oocyte during in vitro spontaneous maturation, suggesting that MOS is located upstream of MAP kinase and is required for MAP kinase activation. MOS/MAP kinase is not necessary for GVBD and PB1 extrusion, although it is involved in microtubule organization [14, 47]. However, these data are achieved using the in vitro spontaneous maturation model. They cannot rule out the possibility that in vivo, mouse oocytes express a redundant activity, such as through the RAS/RAF pathway. Under the stimulation of preovulatory gonadotropin surge, this alternative pathway may compensate in the Mos-/- mice for the lack of MOS activity and induce the activation of MAP kinase and oocyte meiotic resumption. Although it has been reported that RAF is not active during the spontaneous meiotic resumption in Mos-/- oocytes, suggesting it is not involved in oocyte meiotic resumption [14], it should be noted that no evidence has been provided that RAF or MAP kinase is not involved in gonadotropin-induced oocyte meiotic resumption both in vitro or in vivo. In the present report, irrespective of MOS or RAF, we used specific inhibitors of MAPKK, PD98059 and U0126, to examine the role of MAP kinase in FSH-induced mouse oocyte meiotic resumption. It has been reported that PD98059, at a concentration of 50 µM, was ineffective in inhibiting a variety of other kinases both in vivo and in vitro [48]. U0126, a new generation of specific MAPKK inhibitor, has been reported to be effective at lower concentrations than PD98059. The 50% inhibitory concentration (IC50) against a constitutively active MAPKK in vitro was 0.07 µM for U0126 and 10 µM for PD98059, although 10 µM U0126 was required to inhibit MAP kinase phosphorylation completely in cultured cells [49]. The inhibition of FSH-induced oocyte meiotic resumption by these two inhibitors indicates that the MEK-MAP kinase pathway is implicated in FSH-induced oocyte meiotic resumption. This is further reflected by evidence that with FSH stimulation, active MAP kinase was detected prior to GVBD in both the oocytes and the cumulus cells, and the activation of MAP kinase was inhibited by U0126.

Because there are no FSH receptors on the oocyte membrane, the meiotic stimulatory effect of FSH is through its binding with the receptors on the cumulus cells [34]. In vitro, cumulus cells play an important role in FSH-induced oocyte meiotic resumption. Under the stimulation of FSH, epidermal growth factor, or cAMP analogues, cumulus cells of mouse COC can generate a kind of positive stimulus that acts on the oocyte and induces the resumption of meiosis [8, 28, 50, 51]. Further investigation indicates that this kind of positive stimulus might be the recently reported meiosis-activating sterols (MAS) [28, 52]. However, its potential role as a physiological meiotic inducing substance remains inconclusive [53, 54]. A gap junction-transmitted positive signal produced by cumulus cells of mouse COC under the stimulation of FSH might play a potential role in meiotic induction [55]. It has been reported that priming mouse COC with FSH for 0.5–2 h is enough to trigger the cumulus cells to generate a kind of meiosis-inducing signal(s) that acts on the oocyte and induces the resumption of meiosis [28]. Interestingly, in our study, we found that FSH could also activate MAP kinase within the cumulus cells at a similar time. It is possible that MAP kinase activation within the cumulus cells is responsible for FSH to trigger the cumulus cells to produce this meiosis-inducing signal(s). Protein kinase A, protein kinase C, and calcium might be involved in FSH-induced MAP kinase activation in the cumulus cells, because these three signalling pathways have been reported to be involved in FSH-induced oocyte meiotic resumption [32, 51, 56].

On the other hand, in our study, FSH-induced oocyte meiotic resumption may be through the MEK-MAP kinase pathway in the oocytes, because within the oocytes, active MAP kinase could also be detected prior to GVBD. Indeed, some reports have already shown that microinjection of MOS or MAPKK (MEK) RNA into immature mouse oocytes could fully activate MAP kinase in the absence of MPF activation and induce the oocytes to override the meiotic arrest of PDE inhibitor and resume meiosis [23]. In our study, the activation of MAP kinase within the oocytes might be induced by the cumulus cell-generated meiosis-inducing signal(s). This signal(s) might be the recently reported MAS [52]. However, more recently, data from two groups published at the same time failed to support MAS as a natural meiotic inducer [53, 54]. The physiological involvement of MAS in oocyte meiotic resumption is still questionable. Other kinds of signal(s) such as intracellular calcium mobilized within the oocytes might also be involved in this process, because it has been reported that intracellular calcium is involved in FSH-induced oocyte meiotic resumption in the mouse [32] and pig [56], and chelation of the intracellular calcium within the oocytes can inhibit MAP kinase activation and oocyte meiotic resumption in bovine [57].

In our study, we found that MAP kinase within the cumulus cells is activated earlier than in oocytes. It was detected at 1 h of FSH stimulation, and maximum activation could be detected between 4 and 8 h. Furthermore, unlike in oocytes, MAP kinase activity within the cumulus cells decreased quickly after 12 h of FSH stimulation and disappeared during the following culture period from 16 to 24 h, exhibiting dephosphorylation of MAP kinase. This phenomenon explains why no MAP kinase activity within the cumulus cells could be detected at 20 h of FSH stimulation in the first experiment. In the present study, we could not decipher the role of MAP kinase dephosphorylation within the cumulus cells during FSH-induced mouse oocyte meiotic resumption. This is an intriguing question in need of further investigation. Under FSH stimulation, some oocyte-derived paracrine regulators might be involved in this process [58].

Taken together, our data show that under the present experimental conditions, the MEK-MAP kinase pathway is involved in FSH-induced oocyte meiotic resumption. MAP kinase is activated prior to GVBD in both oocytes and cumulus cells, suggesting that the MEK-MAP kinase pathway in both types of cells might share the same responsibility for FSH-induced oocyte meiotic resumption. Further studies are required to focus on the involvement of MAP kinase in gonadotropin-induced oocyte meiotic resumption in vivo, as well as to identify in which type of cells MAP kinase activation is actualy involved in this process.

ACKNOWLEDGMENTS

We are greatful to Drs. John J. Eppig and Ieuan M. Joyce at the Jackson Laboratory for critical reviewing the manuscript.

FOOTNOTES

First decision: 8 December 2000.

1 This research was supported by grants from the Ihel Foundation and Kort China-Israel Cooperative Foundation to H.B. Back

2 Correspondence. FAX: 972 3 5344766; breith{at}mail.biu.ac.il Back

3 Current address: The Jackson Laboratory, Bar Harbor, ME 04609. Back

Accepted: March 12, 2001.

Received: November 3, 2000.

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