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Biology of Reproduction 63, 658-665 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

A Role for the MEK-MAPK Pathway in Okadaic Acid-Induced Meiotic Resumption of Incompetent Growing Mouse Oocytes1

Corinne de Vantéry Arrighi2,a, Aldo Campanaa, and Sabine Schorderet-Slatkinea

a Clinique de Stérilité et d'Endocrinologie Gynécologique, Département de Gynécologie et Obstétrique, Maternité, Hôpitaux Universitaires de Genève, 1211 Genève 14, Switzerland

ABSTRACT

Fully grown competent mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment, in contrast to growing incompetent oocytes, which remain blocked in prophase I. The cell cycle regulators, maturation promoting factor (MPF; [p34cdc2/cyclin B kinase]) and mitogen-activated protein (MAP) kinases (p42MAPK and p44MAPK), are implicated in meiotic competence acquisition. Incompetent oocytes contain levels of p42MAPK, p44MAPK, and cyclin B proteins that are comparable to those in competent oocytes, but their level of p34cdc2 is markedly lower. Okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, induces meiotic resumption of incompetent oocytes. The kinetics and the percentage of germinal vesicle breakdown depends on whether or not oocytes have been cultured before OA treatment. We show that the fast kinetics and the high percentage of germinal vesicle breakdown induced by OA following 2 days in culture is neither the result of an accumulation of p34cdc2 protein, nor to the activation of MPF in incompetent oocytes, but rather by the premature activation of MAP kinases. Indeed, a specific inhibitor of MAPK kinase (MEK) activity, PD98059, inhibits activation of MAP kinases and meiotic resumption. Altogether, these results indicate that the MEK-MAPK pathway is implicated in OA-induced meiotic resumption of incompetent mouse oocytes, and that the MEK-MAPK pathway can induce meiotic resumption in the absence of MPF activation.

kinases, meiosis, oocyte development, phosphatases

INTRODUCTION

During oogenesis, mouse oocytes enter the prophase of the first meiotic division and remain arrested at the dictyate stage of prophase I (secondarily decondensed diplotene chromosomes) or at the germinal vesicle (GV) stage. Shortly after birth, they undergo a period of extensive growth, and their diameter increases from about 20 to 80 µm. They are transcriptionally and translationally active [1, 2]. Toward the end of this growth phase, when they reach about 80% of their full size, oocytes acquire meiotic competence, and they will spontaneously resume meiosis in vitro when released from their follicular environment [3–5]. Mouse oocytes have been classified as incompetent or competent according to their respective inability or ability to spontaneously resume meiosis in vitro. Acquisition of meiotic competence occurs in two steps: First, the oocytes become competent to undergo germinal vesicle breakdown (GVBD) and to assemble a metaphase I (MI) spindle with condensed chromosomes (G2- to MI-phase transition); and second, they later acquire the ability to progress until metaphase II (MII) [4, 6]. Isolation of the first wave of developing oocytes at various days following birth yields a relatively synchronous population [7]. Oocytes from 12-day-old mice are contained within preantral follicles, are in mid-growth phase, and are incompetent of meiotic resumption. Although little is known about the mechanisms involved, meiotic competence acquisition depends on follicular cells [8, 9] and on an intrinsic autonomous program of differentiation (internal clock) [10, 11].

Meiotic maturation is controlled by the cytoplasmic M-phase or maturation promoting factor (MPF), the key regulator of both mitotic and meiotic cell cycles, and the mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERK), which have been implicated in many signal transduction pathways. MPF is a complex of cyclin B and p34cdc2 activated through the dephosphorylation of threonine 14 and tyrosine 15 residues of p34cdc2 by Cdc25 phosphatase and phosphorylation of threonine 161 of p34cdc2 by cyclin-dependent kinase (CDK)-activating kinase (CAK). MPF is maintained inactivated by the Wee1 kinase, Myt1 kinase, and inhibitors (CDKIs). Moreover, upstream kinases (Polo-like kinases) and phosphatases (protein phosphatase 2 [PP2A]) are also implicated in MPF regulation [12–18]. MAP kinases are serine/threonine kinases activated by phosphorylation of threonine and tyrosine residues that are activated by a wide variety of oncogenes and extracellular stimuli. MAPKs participate in the signaling cascade downstream of growth factor/cytokine receptors, Ras, Raf, and MAPK kinase (MEK). Activation of MAPKs is more complicated than a simple linear pathway, and evidence exists for multiple, temporally distinct pathways converging on MAP kinases that are differentially utilized by various stimuli and cell types. MEK-dependent and MEK-independent pathways exist that regulate activation of MAPKs [19, 20]. In the oocyte system, MAPKs are activated through a pathway that is believed to involve the p39mos kinase and probably MEK or through the Ras-Raf-MEK pathway [21–30]. MEK is a dual-specificity kinase that phosphorylates MAP kinases on threonine and tyrosine residues in the catalytic domain [31, 32]. MAP kinases are inhibited by PP2A, a protein tyrosine phosphatase, and/or Tyr/Thr-protein phosphatases (CL100/3CH134/MKP-1, MKP-3) [33–35]. Activated MAPKs may directly or indirectly regulate some proteins that control MPF activity, such as Cdc25 phosphatase, Wee1 kinase, Myt1 kinase, and CAK [36–39]. MAPKs have thus been implicated in the cascade leading to MPF activation, but an alternative pathway, in which MAPKs are activated by MPF, has also been proposed [40, 41]. p42MAPK (ERK2) and p44MAPK (ERK1) proteins [42–45] are activated during meiotic resumption of Xenopus [26, 46, 47] and mouse [48–51] oocytes. We and others demonstrated that p34cdc2, in contrast to cyclin B, p42MAPK, and p44MAPK, is less abundant in incompetent than in competent mouse prophase I oocytes, and that meiotic competence acquisition correlates with the presence of high levels of p34cdc2 protein [52–55].

The inhibition of serine/threonine phosphatases 1 and 2A by okadaic acid (OA) [56, 57] induces meiotic resumption of oocytes of many species, including both competent [48, 51, 58–63] and incompetent [52–54, 60, 64–66] mouse oocytes. Incompetent oocytes that have been freshly picked up respond to OA with delayed and asynchronous kinetics of meiotic resumption (GVBD) [54, 60, 61, 65]; however, if incompetent oocytes are first cultured for 2 days and then treated with OA, their response is increased as demonstrated first by the fast and synchronous kinetics of meiotic maturation (GVBD) and, second, by the high percentage of oocytes resuming meiosis [61, 65]. In both cases, incompetent oocytes are arrested near MI because chromosomes are not aligned on the metaphase plate and are slightly decondensed [54, 67–69].

The aim of the present study was to determine: 1) the morphological (chromatin) and biochemical (p34cdc2, p42MAPK, and p44MAPK) differences between freshly picked-up and 2-day-cultured incompetent oocytes and 2) the role of MPF and MAPKs in the fast kinetics of OA-induced meiotic resumption of 2-day-cultured incompetent oocytes. Taken together, our results indicate that the MEK-MAPK pathway is implicated in OA-induced meiotic resumption of incompetent mouse oocytes.

MATERIALS AND METHODS

Oocyte Collection and Culture

All procedures involving mice were conducted in accordance with Swiss Office Veterinaire Cantonal authorizations and regulations concerning the use and care of experimental animals.

Fully grown meiotically competent oocytes were harvested from the ovaries of 20- to 28-day-old randomly bred Swiss albino mice. The follicles were punctured with fine forceps in minimal essential medium (MEM) with Earles salts (Gibco BRL, Paisley, UK) [60, 67]. The medium was supplemented with 200 µM phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma Chemical Company, St. Louis, MO) to prevent the spontaneous resumption of meiosis [70]. Oocytes were freed of attached cumulus cells.

Growing, meiotically incompetent oocytes were harvested from ovaries of 12-day-old randomly bred Swiss albino mice. Ovaries were excised and then dissected in medium 199 (M199) with Earles salts (Gibco BRL) [67]. Oocytes were enzymatically dissociated from follicular cells in a medium containing 250 U/ml DNase I (Sigma), 3.5 U/ml trypsin (Sigma), and 59 U/ml collagenase (Worthington, Freehold, NJ).

Oocytes were cultured in a drop of medium (20 oocytes/50 µl) at 37°C in a humidified atmosphere of 5% CO2 under mineral oil (Sigma).

Oocyte Treatment with Okadaic Acid, PD98059, or Cycloheximide

Incompetent and competent oocytes were incubated in an OA-containing M199 medium (Gibco) at a final concentration of 1 µM for 2 h, washed, and then transferred to a control medium.

PD98059 (Calbiochem, San Diego, CA), an inhibitor of MEK, was directly dissolved from DMSO stock into M199 medium at a final concentration of 10, 50, or 100 µM. This MEK inhibitor was present 1 h before (preincubation), during, and following OA treatment. Control incubation contained PD98059 alone. The maximum concentration of PD98059 did not affect cell viability and did not induced meiotic resumption.

Cycloheximide (Sigma), was directly dissolved into M199 medium at a final concentration of 100 µg/ml. This protein synthesis inhibitor was present during the entire incubation time.

Western Immunoblotting

Oocytes were lysed in 10 µl of lysis buffer [71]. The samples were subjected to 10%–12% long (Figs. 2 and 4B) or short (Fig. 4, A, C, and D; Fig. 6) SDS-PAGE [72]. Proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) in cold buffer (25 mM Tris, 192 mM glycine, 25% methanol, 0.025% SDS) using a Bio-Rad transfer apparatus. p34cdc2>, p42MAPK, p44MAPK, phospho-p44/p42 MAP kinase, and phospho-specific MEK1/2 were detected by probing the membranes with, respectively, a monoclonal anti-p34cdc2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), a polyclonal anti-ERK2 (p42MAPK) antibody (Santa Cruz Biotechnology), a polyclonal anti-ERK1 (p44MAPK) antibody (Santa Cruz Biotechnology), a polyclonal anti-phospho-p44/p42 MAP kinase (ERK1/2) antibody (New England Biolabs, Beverly, MA), and a polyclonal anti-phospho-specific MEK1/2 antibody (New England Biolabs) using a Blotting Chemiluminescence Detection Kit (Amersham, Buckinghamshire, UK) according to the manufacturer's instructions. For reprobing, the blots were stripped of bound antibodies by washing in a stripping buffer [54] at 50°C for 30 min. Equal numbers of incompetent oocytes were subjected to immunoblotting.



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  		FIG. 2. Immunodetection of p34cdc2, p42MAPK, and p44MAPK proteins in growing incompetent oocytes before (0 h) and following 2 days (48 h) of culture. Blots were probed with anti-p34cdc2 (A) or anti-ERK2 (p42MAPK) and anti-ERK1 (p44MAPK) (B) antibodies. Equal number of oocytes (100 for A, 200 for B) were loaded per lane. The experiment was performed 6 times with similar results



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  		FIG. 4. Premature activation of MAPKs in growing incompetent oocytes transiently exposed to OA following 2 days of culture. A) Incompetent oocytes were lysed following OA treatment but before GVBD (OA) and processed for histone H1 and MAP double kinase assays; nontreated incompetent prophase oocytes (C) served as controls. Twenty oocytes were loaded per lane. The experiment was performed 3 times with similar results. B, C, D) Oocytes cultured during 2 days and then transiently treated with OA were lysed before GVBD (OA) and processed for Western immunoblotting; nontreated incompetent prophase oocytes (C) served as controls. Blots were probed with anti-ERK2 (p42MAPK), anti-ERK1 (p44MAPK; B, D), anti-phospho-p44/p42 MAP kinase (p44MAPK-P, p42MAPK-P), and anti-phospho-specific MEK1/2 (MEK1/2-P; C) antibodies. Two hundred (B) and 120 (C, D) oocytes were loaded per lane. The experiment was performed 3 times with similar results



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  		FIG. 6. Inhibition of MAPK activation in growing incompetent oocytes transiently exposed to OA in the presence of an MEK inhibitor, PD98059. Incompetent oocytes cultured during 2 days (48 h) were treated 1 h before OA treatment with the MEK inhibitor, PD98059 (100 µM; PD). OA-treated incompetent oocytes served as controls (C). The blot was sequentially probed with anti-phospho-p44/p42 MAP kinase (p44MAPK-P, p42MAPK-P), and anti-phospho-specific MEK1/2 (MEK1/2-P; A) and then with anti-ERK2 (p42MAPK) and anti-ERK1 (p44MAPK; B) antibodies. Equal numbers of oocytes (200) were loaded per lane. The experiment was performed 4 times with similar results

Histone H1 and MAPK In Vitro Assays

Oocytes were washed and then lysed in homogenization buffer [51, 54]. The reaction was started by the addition of 10 µl of a solution containing 10 mM MgCl2; 20 µM protein kinase inhibitor (PKI; Sigma); 0.2 mM ATP (Sigma); 20 µg/ml each of pepstatin, chymostatin, and aprotinin; 40 µg/ml leupeptin; 0.4 mM sodium-orthovanadate; 5 µCi [{gamma}-32P]ATP (Amersham); 10 µg histone H1 (Boehringer Mannheim, Mannheim, Germany); and 10 µg myelin basic protein (MBP; Sigma). Reactions were performed at 37°C for 20 min and stopped by boiling for 3 min after addition of 20 µl of a twofold concentrated electrophoresis sample buffer [72]. The samples were subjected to 15% SDS-PAGE. The gels were stained with Coomassie brillant blue R250 (Fluka, Buchs, Switzerland) and dried on filter paper. The bands corresponding to histone H1 and MBP were excised from the dried gels. Radioactivity was measured by liquid scintillation counting.

Immunofluorescence Staining and Microscopy

Oocytes were fixed and extracted in a microtubule-stabilizing buffer [54, 60], washed in PBS (Sigma) containing 0.1% BSA (PBS/BSA), and incubated with a mouse anti-chick {alpha}-tubulin antibody (Amersham) diluted 1:2000. They were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (IgG; Biogenzia Lemania, Lausanne, Switzerland) diluted 1:20, washed again, incubated in 10 µg/ml of Hoechst dye 33258 (Fluka) for DNA staining, and finally mounted in Mowiol [73] and viewed on a Leitz epifluorescence microscope [54, 60].

Chromosome Preparation

The air-drying method for chromosome preparation was used [74]. Briefly, OA-induced incompetent oocytes at or near MI were soaked in a hypotonic 1% sodium citrate (Sigma) physiological solution Ringer (Sigma) for 10 min at room temperature. A microdrop of this solution together with the oocytes was placed on a grease-free glass slide. The oocytes were then fixed by expelling a few drops of acetic acid-ethanol 1:3 on the microdrop and then air-dried. Finally, the preparation was stained with 2% toluidine blue for a few minutes, washed under tap water, dried, and observed with a microscope with a x100 objective. Immersion oil was applied directly onto the slide.

RESULTS

Effect of Culture on Meiotic Competence Acquisition of Incompetent Mouse Oocytes

Our aim was to elucidate whether the difference in the kinetics of meiotic resumption induced by OA between freshly picked up (26% GVBD by 24 h) and 2-day-cultured (94% GVBD by 4 h) incompetent oocytes is the result of a sequential change in the organization of nuclear chromatin and nucleolar-associated chromatin; or to the accumulation or activation of cell cycle proteins p34cdc2, p42MAPK, and p44MAPK associated with oogenesis progression and expression of meiotic competence; or both.

Our results show that GV chromatin of freshly picked-up incompetent oocytes correspond to the pattern of staining with Hoechst, known as stages I and II according to Mattson et al. [75] (Fig. 1A), whereas that of 2-day-cultured incompetent oocytes correspond to stages III and IV (Fig. 1B). In stages I-II, GV contains several chromatin foci associated with the nucleolar periphery, whereas in stages III-IV, GV exhibits a partial or total rim of chromatin staining at the nucleolar surface. In addition, in stages III-IV, GV exhibits a more reticular or fibrillar chromatin pattern throughout the nucleoplasm. The transition from stages I-II to III-IV during the culture shows that incompetent oocytes progress toward meiotic competence acquisition.



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  		FIG. 1. Germinal vesicle chromatin in growing incompetent oocytes before and following 2 days of culture. Incompetent prophase oocytes were fixed before (A) or following (B) 2 days in culture. Oocytes were stained with Hoechst dye (A, B). Nucleolus (n), chromatin foci (arrow) and chromatin nuceolar riming (arrowhead) are shown. The experiment was performed 3 times with similar results. Magnification x1000

Because it was previously shown that p34cdc2, p42MAPK, and p44MAPK are implicated in meiotic resumption of mouse oocytes, we compared the amount of these cell cycle proteins in freshly picked-up and 2-day-cultured incompetent oocytes by Western immunoblotting. As shown in Figure 2, p34cdc2, p42MAPK, and p44MAPK were present in similar amounts in both conditions. In some immunoblots, p34cdc2 was even present in a lesser amount following the 2 days in culture (data not shown). These proteins were not activated during the culture because p34cdc2 is present in the three migrating bands (Fig. 2A) and there is no upward shifts in migration of p42MAPK and p44MAPK from phosphorylation (Fig. 2B). These results show that the difference in the kinetics of GVBD is neither the result of the accumulation or activation of p34cdc2, nor to MAPKs.

Partial Meiotic Resumption of Incompetent Oocytes by Okadaic Acid

Incompetent oocytes cultured for 2 days and then treated with OA resumed meiosis and underwent GVBD, but did not extrude the first polar body. We consequently analyzed the spindle and the chromosomes. They often assembled an elongated and multipolar spindle (Fig. 3A) and the chromosomes were dispersed throughout the spindle instead of being aligned on the metaphase plate (Fig. 3B), as previously described by us [54, 60, 67] for OA-treated freshly picked-up incompetent oocytes or for p34cdc2 and cyclin B1 mRNA microinjected-incompetent oocytes. They are thus blocked near or at MI. Because chromosomes seemed to be less condensed (longer), we analyzed them by the air-drying method. As shown in Figure 3C, chromosomes stained with toluidine blue are present as bivalents, but compared with chromosomes of competent OA-induced (1 µM for 2 h) MI oocytes (Fig. 3D), they are insufficiently condensed and present differences in the degree of condensation within the same oocyte, suggesting an arrest in pro-MI.



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  		FIG. 3. Arrest in prometaphase I of growing incompetent oocytes transiently exposed to OA following 2 days of culture. A, B) Incompetent oocytes cultured during 2 days and then transiently treated with OA were fixed 20 h following OA treatment. Incompetent oocytes were stained by indirect immunofluorescence using an anti-{alpha}-tubulin antibody (A) or with Hoechst dye (B). The spindle is abnormal (A) and the chromosomes are dispersed throughout the spindle (B) instead of being aligned on the metaphase plate. The experiment was performed 3 times with similar results. Magnification: x1200 (A) and x1000 (B). C) OA-treated incompetent and (D) competent oocytes were processed for chromosome analysis (toluidine blue staining). Chromosomes form bivalents (C, D) but those of incompetent oocytes are less condensed (longer) than competent ones. The experiment was performed 3 times with similar results. Magnification: x1000

Premature Activation of the MEK-MAPK Pathway

Our aim was to elucidate whether OA-induced meiotic resumption of incompetent oocytes cultured for 2 days was mediated through the MPF or the MEK-MAPK pathway. We thus analyzed the activity of these proteins by in vitro kinase assays, Western immunoblotting, or both.

Incompetent oocytes were cultured for 2 days and then treated with OA (1 µM for 2 h). Following OA treatment but before GVBD, histone H1 kinase was only marginally activated (mean of 1.5-fold, n = 3), whereas MAPK was highly activated (mean of 8.5-fold, n = 3; Fig. 4A) in incompetent oocytes. Thereafter, histone H1 kinase activity increases slightly up to MI (data not shown). The histone H1 and MAPK assays were linear with respect to time (for up to 40 min) and number of MII-arrested oocytes analyzed (up to 20; data not shown). The activation of MAPK following OA treatment was confirmed by Western immunoblotting using polyclonal antibodies anti-ERK2, anti-ERK1, and anti-phospho-p44/p42 MAP kinase. Following OA treatment, p42MAPK and p44MAPK were phosphorylated and thus activated, as revealed by upward shifts in migration (Fig. 4B) or appearance of the bands (Fig. 4C). MEK, the MAPK kinase, was also phosphorylated and thus activated, as detected by an anti-phospho-specific MEK1/2 (Fig. 4C). MEK, p42MAPK, and p44MAPK were already slightly phosphorylated after 1 h of OA-treatment (data not shown). Figure 4D shows the presence of equal amounts of p42 and p44MAPK, validating the differences observed for phosphorylated MAPK and MEK in control (C)- and OA-treated oocytes (Fig. 4C).

Our results indicate that MEK and MAP kinases are activated before MPF in incompetent oocytes treated by OA following 2 days in culture.

Inhibition of MAPK Activation and Meiotic Resumption by PD98059

We used an inhibitor of MEK, PD98059, as a pharmacological tool to study the involvement of MAP kinases in the induction of meiotic resumption of incompetent oocytes treated by OA following 2 days in culture. We consequently treated incompetent oocytes cultured for 2 days with 100 µM PD98059 for 1 h before, during, and following OA treatment. As expected, PD98059 inhibited meiotic resumption (Fig. 5) and phosphorylation of p42MAPK and p44MAPK (Fig. 6). Nevertheless, meiotic inhibition is transient and not fully reversible because a lesser proportion of oocytes resume meiosis (mean of 50% GVBD after 1 day; Fig. 5). Lower concentrations of PD98059 (10 and 50 µM) resulted in a proportionally lower yield of meiotic inhibition (Fig. 5). p42MAPK and p44MAPK phosphorylation and thus activation were inhibited, as revealed by Western immunoblotting using the anti-phospho-p44/p42 MAP kinase antibody (Fig. 6A). The incompetent oocytes, which overcame the presence of the MEK inhibitor by resuming meiosis (GVBD) contained activated p42MAPK and p44MAPK (data not shown). MEK appeared phosphorylated in the presence of PD98059, as detected by a polyclonal antibody anti-phospho-specific MEK1/2 (Fig. 6A). Detection of total p42MAPK and p44MAPK proteins were used as a control in equal amounts in all conditions (Fig. 6B). We also tested the effect of cycloheximide (100 µg/ml) on OA-induced meiotic resumption of 2-day cultured incompetent oocytes because activation of MAP kinases required protein synthesis. No oocytes underwent GVBD (0%) for up to 1 day (data not shown), indirectly confirming the implication of MAP kinases in OA-induced meiotic resumption.



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  		FIG. 5. Delayed kinetics of meiotic resumption of growing incompetent oocytes transiently exposed to OA in the presence of the MEK inhibitor, PD98059. Incompetent oocytes cultured during 2 days (48 h) were treated 1 h before OA-treatment with PD98059, at concentrations of 10, 50, and 100 µM (triangle, square, cross). PD98059-nontreated but OA-treated incompetent oocytes (square) served as controls. The proportion (%) of incompetent oocytes having resumed meiosis (i.e., having undergone GVBD) was scored at different times. This figure represents the data from 1 out of 4 experiments with similar results. Statistical significance was determined using multiple analysis of variance with a Fisher test: for PD100 µM + OA vs. OA at 2 h 30 min, 3 h 30 min, and 4 h 30 min, P < 0.007, at 5 h 30 min and 20 h, P < 0.04; for PD50 µM + OA vs. OA at 2 h 30 min, P < 0.02, and for PD10 µM + OA vs. OA at 2 h 30 min and 3 h 30 min, P < 0.05. For all other points, results were not found to be statistically significant

Together, our results indicate that OA induces meiotic resumption of incompetent oocytes through the precocious activation of the MEK-MAPK pathway.

DISCUSSION

We and others previously showed that the acquisition of meiotic competence at the end of oocyte growth appears to be associated with 1) the translational activation of dormant p34cdc2 mRNA, resulting in an abrupt increase in the concentration of p34cdc2 protein; and 2) the post-translational modification of cyclin B1 [52, 54, 55, 67]. The MAPK pathway is also implicated in meiotic competence acquisition [53, 54, 65–67]; however, the implication of the MAPK pathway in the induction of meiosis in incompetent mouse oocytes has not been fully investigated.

The fast and synchronous meiotic resumption of oocytes cultured for 2 days before OA treatment could be explained by one or a combination of three factors. First, the reorganization of chromatin, indicating the progression of oocytes toward meiotic competence acquisition; second, the accumulation of p34cdc2 and activation of MPF; and third, the activation of the MAPK pathway. We thus investigated these possibilities.

We observed the reorganization of chromatin (nucleolar riming) in the GV of oocytes cultured for 2 days. Incompetent oocytes thus progressed from stages I-II to III-IV as described by Mattson et al. [75], showing that cultured incompetent oocytes progressed toward meiotic competence acquisition. Indeed, it is known that this sequential alteration in chromatin organization occurs before meiotic competence acquisition [75–77]. Nevertheless, incompetent oocytes cultured for 2 days did not acquire the capacity of spontaneous meiotic resumption.

Our results showed that p34cdc2, which is present in low levels in incompetent, freshly picked-up oocytes [54], does not accumulate during the 2 days of culture. This finding is different from that of Chesnel and Eppig [52] who showed that the total amount of p34cdc2 of incompetent oocytes increased during culture. They observed a threefold increase of p34cdc2, an amount still lower than that in competent oocytes. This discrepancy may be explained by the age of mice used to pick up oocytes (15- instead of 12-day-old females) and by the number of days incompetent oocytes have been cultured (3 days instead of 2). However, incompetent oocytes did not spontaneously resume meiosis. Our results and those of Chesnel and Eppig [66] showed that p42MAPK and p44MAPK did not accumulate and are not activated during the culture period.

We showed that incompetent oocytes cultured for 2 days and then treated with OA resume meiosis with premature MAPK activation, thus confirming the results of Chesnel and Eppig [66]. Indeed, MAP kinases are activated before GVBD, whereas histone H1 kinase is not activated. Our results showed that MEK is present in incompetent mouse oocytes, phosphorylated, and thus activated following OA treatment.

Activation of the MAPK pathway without MPF has been previously described in competent mouse, rat, and Xenopus oocytes. OA treatment of competent mouse oocytes blocked in prophase I (GV stage) by IBMX induces GVBD and MI entry with premature MAPK activation (before GVBD) in the absence of histone H1 kinase activation [51]. MAPK can also be activated in competent mouse oocytes blocked in prophase I by IBMX by injecting either Mos or MEK mRNA. Meiosis I is then significantly prolonged, and MAPK becomes fully activated in the absence of MPF [78]. In Xenopus oocyte extracts, constitutively active Mos also induced activation of MAPK without MPF [22]. Chesnel et al. [30] explained the incapacity of Xenopus stage-IV oocytes to activate MPF in response to insulin primarily by their inability to activate Ras and to stimulate Mos synthesis and, secondarily, by a deficiency in the mitogenic pathway that connects MAPK to MPF activation. Indeed, in contrast to mouse incompetent oocytes, injection of constitutively active Raf mRNA or Ras protein in incompetent Xenopus stage-IV oocytes resulted in activation of MAPK without inducing GVBD, suggesting that the Ras-Raf-MEK-MAPK cascade was functional, but that MAPK activation alone was not sufficient for meiotic resumption [30]. In rat oocytes, OA induces only partial MPF activation, whereas activation of MAPK was accelerated and began 1 h after GVBD; therefore, 2 h earlier than in control oocytes [79].

In order to confirm the implication of the MAPK pathway in meiotic resumption of incompetent oocytes cultured for 2 days and then treated with OA, we used an inhibitor of MEK, PD98059. PD98059 selectively blocks the activity of MEK by inhibiting the activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells [80–83]. Our results showed that this inhibitor prevented activation of MAPKs (p42MAPK and p44MAPK) and meiotic resumption (GVBD). Exposure of incompetent oocytes to PD98059 did not affect the overall contents of MAPKs but resulted in a loss of the dually phosphorylated forms of MAPKs, corresponding to active p42MAPK and p44MAPK. Nevertheless, meiotic inhibition is transient and not fully reversible because a lesser proportion of oocytes finally resume meiosis. This transient effect may be the result of the presence of both OA and MEK inhibitor acting on the same target, MEK. OA may overcome the effect of the MEK inhibitor by activating MEK through the inhibition of PP2A [84].

In our system, following OA treatment, MEK is phosphorylated in the presence of the MEK inhibitor, PD98059, as detected by a polyclonal antibody anti-phospho-specific MEK1/2. It has been demonstrated that PD98059, a reversible inhibitor of MEK, prevents the activation and phosphorylation of MEK by Raf or MEK kinase in vitro and in vivo [80, 81]. The fact that in our conditions MEK is phosphorylated may be due to the partial reversibility of the inhibition of meiosis induced by PD98059, the time-dependent antagonist effect of OA and PD98059 on the same target, MEK, or both. In Chaetopterus oocytes, exposure to PD98059 inhibited phosphorylation of MAPKs, but in contrast to mouse oocytes, it did not block GVBD [85]. In Xenopus oocytes, the microinjection of an inhibitory synthetic unphosphorylated MAPK peptide or the exposure to PD98059 blocked the activity of MAPK and prevented the progesterone-induced oocyte maturation. As for mouse oocytes, PD98059 failed to completely prevent the activation of MAPK and meiotic maturation of oocytes [86, 87]; these researchers suggested that a little residual activation of MEK induced by progesterone in the oocytes treated with MEK inhibitors may, in the long run, bring about the activation of MAPK. On the other hand, in Swiss 3T3 cells, PD98059 inhibited the activation of MAPK, but the extent of inhibition depended on how potently Raf and MEK were activated by any particular agonist [80].

Taken together, these results show that depending on the experimental system and conditions, activation of MAP kinases may or may not be sufficient to induce GVBD, and that activation of MAPKs and p34cdc2 can occur on independent, parallel pathways, or on the same pathway. It is suggested that imbalanced coordination between protein kinases and protein phosphatases determines the cellular responses. Our results suggest that incompetent oocytes cultured for 2 days and then treated with OA, an inhibitor of phosphatases 1 and 2A, resume meiosis with fast kinetics of GVBD through the MEK-MAPK pathway.

ACKNOWLEDGMENTS

We thank V. Laplana for technical help and J.-F. Arrighi for a critical review of the manuscript.

FOOTNOTES

First decision: 20 October 1999.

1 This work is supported by HUG. Back

2 Correspondence: Corinne de Vantéry Arrighi, Clinique de Stérilité et d'Endocrinologie Gynécologique, Département de Gynécologie et Obstétrique, Maternité, Hôpitaux Universitaires de Genève, 32, bd de la Cluse, 1211 Genève 14, Switzerland. FAX: 41 22 382 44 24; corinne.devantery{at}hcuge.ch Back

Accepted: March 20, 2000.

Received: September 27, 1999.

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