Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte

Developmental Biology 267 (2004) 265 – 278 www.elsevier.com/locate/ydbio

Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte Sabine Vaur, a Robert Poulhe, b Gilliane Maton, b Yannick Ande´ol, a and Catherine Jessus b,* a

Equipe ‘‘Re´gulations post-transcriptionnelles et de´veloppement pre´coce’’, Laboratoire de Biologie du De´veloppement, UMR-CNRS 7622, Universite´ Pierre et Marie Curie, 75252 Paris cedex 05, France b Equipe ‘‘Biologie Cellulaire de l’ovocyte’’, Laboratoire de Biologie du De´veloppement, UMR-CNRS 7622, Universite´ Pierre et Marie Curie, 75252 Paris cedex 05, France Received for publication 25 July 2003, revised 1 December 2003, accepted 3 December 2003

Abstract Activity of Cdc2, the universal inducer of mitosis, is regulated by phosphorylation and binding to cyclin B. Comparative studies using oocytes from several amphibian species have shown that different mechanisms allow Cdc2 activation and entry into first meiotic division. In Xenopus, immature oocytes stockpile pre-M-phase promoting factor (MPF) composed of Cdc2-cyclin B complexes maintained inactive by Thr14 and Tyr15 phosphorylation of Cdc2. Activation of MPF relies on the conversion of pre-MPF into MPF by Cdc2 dephosphorylation, implying a positive feedback loop known as MPF auto-amplification. On the contrary, it has been proposed that pre-MPF is absent in immature oocyte and that MPF activation depends on cyclin synthesis in some fishes and other amphibians. We demonstrate here that MPF activation in the axolotl oocyte, an urodele amphibian, is achieved through mechanisms resembling partly those found in Xenopus oocyte. Pre-MPF is present in axolotl immature oocyte and is activated during meiotic maturation. However, monomeric Cdc2 is expressed in large excess over pre-MPF, and pre-MPF activation by Cdc2 dephosphorylation takes place progressively and not abruptly as in Xenopus oocyte. The intracellular compartmentalization as well as the low level of pre-MPF in axolotl oocyte could account for the differences in oocyte MPF activation in both species. D 2004 Elsevier Inc. All rights reserved. Keywords: Cdc2; Cyclin; MAP kinase; Amphibian oocyte; Meiosis

Introduction Fully grown oocytes are arrested at the prophase of meiosis I. In amphibians, re-entry into meiosis, or oocyte meiotic maturation, is triggered by the ovarian hormone progesterone secreted from the follicle cells. Progesterone initiates intracellular signaling pathways, ultimately leading to the activation of the M-phase promoting factor (MPF; Karaiskou et al., 2001). Once activated, MPF triggers the structural events of the first meiotic division: chromosome condensation, nuclear envelope breakdown (known as germinal vesicle breakdown or GVBD), and formation of the

Abbreviations: Cdc, cell division cycle; CDK, cyclin-dependent kinase; GVBD, germinal vesicle breakdown; MAPK, mitogen-activated protein kinase; MPF, M-phase promoting factor; PP, protein phosphatase. * Corresponding author. Equipe ‘‘Biologie Cellulaire de l’ovocyte’’, Laboratoire de Biologie du De´veloppement, UMR-CNRS 7622, Universite´ Pierre et Marie Curie, boıˆte 24, 4 place Jussieu, 75252 Paris cedex 05, France. Fax: +33-1-44-27-34-72. E-mail address: [email protected] (C. Jessus). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.12.002

first meiotic spindle. MPF is the universal key molecule controlling G2/M transition in the eukaryotic cell cycle, irrespective of meiosis and mitosis (Nurse, 1990). The biochemistry of oocyte maturation has been thoroughly studied as it provides a good model system to analyze the molecular mechanisms of MPF activation. MPF is a complex of Cdc2, the protein kinase catalytic subunit, and cyclin B, the protein regulatory subunit. The activity of Cdc2 is controlled by binding to cyclin B and phosphorylation at three highly conserved residues (Coleman and Dunphy, 1994). Phosphorylation on Thr161 is necessary for Cdc2 kinase activation. In contrast, phosphorylation on either Thr14 or Tyr15 dominantly inhibits Cdc2 activation. In the Xenopus immature oocyte, MPF exists as an inactive complex, called pre-MPF, formed of Cdc2 and cyclin B, due to inhibitory phosphorylations on Thr14 and Tyr15 (Dunphy et al., 1988; Gautier et al., 1988, 1989). Activation of MPF induced by progesterone depends on the conversion of pre-MPF into MPF by the Cdc25 phosphatase that directly dephosphorylates Thr14 and Tyr15 residues of Cdc2 (Dunphy and Kumagai, 1991; Gautier et al., 1991).

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This conversion is accelerated by a Cdc25/Cdc2 positive feedback loop, known as the auto-amplification of MPF (Masui and Markert, 1971). This loop explains why injection of either active MPF or Cdc25 causes GVBD in Xenopus oocyte (Masui and Markert, 1971; Rime et al., 1994). It has been shown that the positive feedback involves the Xenopus polo kinase, Plx1, that contributes to Cdc25 activation under Cdc2 control, and the Ser/Thr phosphatase 2A, PP2A, that counteracts Plx1 (Abrieu et al., 1998; Karaiskou et al., 1999). As expected for such loop, either component in the pathway can jump-start the system to cause entry into M-phase. Such molecular mechanism of MPF activation, relying on the existence of a pre-MPF stockpile and a positive feedback loop, contributes to the abruptness of entry into M-phase. Oocytes of two species of the Xenopus genus, Xenopus tropicalis and Xenopus laevis, are extremely similar in their molecular responses to progesterone (Bodart et al., 2002; Stanford et al., 2003). However, an alternative molecular mechanism of MPF activation has been described in oocytes of some fishes and amphibians. First, it has been reported that unlike Xenopus, MPF-induced oocyte maturation in Rana pipiens requires protein synthesis (Schuetz and Samson, 1979). Second, in fishes as goldfish, carp, catfish, and lamprey, and two anoura amphibians, Rana japonica and Bufo japonicus, molecular and biochemical studies have revealed that pre-MPF is absent in immature fullgrown oocytes: Cdc2 is present as a monomer and cyclin B could not be detected at the protein level (Tanaka and Yamashita, 1995). This finding argues that MPF activation in oocytes of these species depends on the formation of complexes between Cdc2 and cyclin B in response to the hormonal maturation trigger. In goldfish oocyte, cyclin B accumulates before GVBD, binds to monomeric Cdc2 that becomes phosphorylated on Thr161, and therefore directly activated. Tyr15 phosphorylation of Cdc2 is not detectable during this process (Yamashita et al., 1995). Therefore, in contrast to Xenopus oocytes, MPF activation in fishes and in Rana and Bufo oocytes relies only on the control of cyclin B translation and its association with monomeric Cdc2, independently of Cdc25 phosphatase. Such molecular control of MPF activation implies that Cdc2 kinase is activated progressively and slowly, in a linear function of cyclin B accumulation rate, in contrast with the abruptness characteristic of the pre-MPF/MPF auto-amplification mechanism. These observations suggest the existence of speciesspecific mechanisms of MPF activation during oocyte maturation, irrespective of the generality of MPF structure and function, and implying that the steroid hormone responsible on meiosis resumption in fishes and amphibians oocytes has distinct targets according to different species: either the control of cyclin B translation or the formation of the starter molecules that fire MPF auto-amplification. The study of different amphibian species might provide new insights on the molecular mechanisms of MPF activation,

depending either on cyclin B translation or on pre-MPF activation. In this article, we analyzed the meiotic maturation of an urodele species, the axolotl Ambystoma mexicanum. Unlike the situation in Xenopus, the migration of the germinal vesicle and its breakdown are externally visible in the axolotl oocyte, as well as the first polar body, and this assists greatly in determining the progress of maturation (Beetschen and Gautier, 1989). Our results show that preMPF is present in the axolotl immature oocyte. Moreover, MPF activation and GVBD can be induced by Cdc25 microinjection or MPF transfer independently of protein synthesis, showing that the auto-amplification feedback loop is functional. However, MPF activation differs from the situation described in Xenopus oocyte: first, monomeric Cdc2 is in large excess when compared to pre-MPF molecules, and second, activation of pre-MPF by Tyr15 dephosphorylation of Cdc2 takes place progressively, and not in an abrupt manner as expected for an auto-amplification mechanism. These observations suggest that although pre-MPF is present and contributes to MPF activation, the auto-amplification mechanism is somehow slowed down in axolotl oocyte, probably by the intracellular compartmentalization of this giant cell.

Materials and methods Oocyte treatments and extracts A. mexicanum adult females were bred and maintained under laboratory conditions. Reagents, unless otherwise specified, were from Sigma. Stage VI oocytes were selected after surgical removal of ovaries from mature adult females and were manually defolliculated with forceps under microscope. They were staged according to Beetschen and Gautier (1989) and kept at 18 – 20jC in Modified Barth HighSalt solution (MBSH) (Gurdon and Wickens, 1983). For in vitro maturation, stage VI oocytes (2 mm in diameter) were incubated in the presence of 1 AM progesterone in MBSH medium. The usual volume of microinjection was 100 nl, except for cytoplasm transfer experiments where 200 nl of cytoplasm from matured oocytes were injected into axolotl stage VI oocytes. Stage VI oocytes were microinjected with the phosphatase inhibitor okadaic acid (ICN, 1 AM intracellular concentration), with in vitro transcribed mouse Mos RNA (20 ng per oocyte), and with either recombinant human Cdc25A phosphatase or human cyclin A proteins (0.05– 0.1 mg/ml intracellular concentration, prepared as described in De Smedt et al., 2002; Frank-Vaillant et al., 1999; Karaiskou et al., 1998, respectively). When mentioned, oocytes were preincubated in the presence of 10 Ag/ ml cycloheximide or 50 AM U0126 (Promega). Oocytes were homogenized at 4jC in 10 Al per oocyte of EB buffer (80 mM h-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, pH 7.3) supplemented with protease inhibitor mixture (Sigma P8340) and centrifuged

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at 15 000  g for 30 min at 4jC. Clear supernatant was used for gel filtration, in vitro experiments, Western blot analysis, or for H1 kinase assays. Gel filtration Supernatants (15 000  g, 200 Al) from 40 oocytes were chromatographed on a Superose 12 gel filtration column (Amersham Biosciences) at 0.5 ml/min in column buffer (EB adjusted to 0.1 M NaCl). Ten fractions of 1 ml were collected and subjected to Western blot or histone H1 kinase assay. In some experiments, fractions 7 and 9 were supplemented by adding yeast recombinant Civ1 protein (0.06 Ag/ Al) and human cyclin A (0.2 Ag/Al), as described (De Smedt et al., 2002). In vitro extracts Axolotl prophase oocyte lysates were incubated at 30jC in the presence of 1 AM okadaic acid (ICN) and/or recombinant Cdc25A (0.05 mg/ml in extract) and an ATP-regenerating system (10 mM creatine phosphate, 80 Ag/ml creatine phosphokinase, 1 mM ATP, 1 mM MgCl2). Samples were collected at the indicated times for Western blot analysis and kinase assays.

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final volume of 50 Al in the presence of 100 AM ATP, 3 ACi of [g-32P]ATP (ICN), 10 mM MgCl2, and 0.3 mg/ml of histone H1. After 30 min at 30jC, the kinase reactions were analyzed by SDS-PAGE and autoradiography. For quantification, the histone H1 bands were excised from the gel, and radioactive phosphate incorporation was determined by liquid scintillation counting. Phosphatase 2A assay Casein (5 mg, Sigma C4765) was phosphorylated by 250 mU of the catalytic subunit of PKA (Sigma P2645) for 2 h at 30jC in the presence of 100 AM ATP and 250 ACi [g32P]ATP. Reaction was stopped by addition of 10 mM EDTA, 30 mM NaF, and 2 mM PPi. Proteins were then precipitated twice at 0jC with an equal volume of 90% saturated ammonium sulfate solution. Free nucleotides were removed by chromatography on Sephadex G25 (Pharmacia). [32P]phosphorylated casein was incubated for 20 min at 30jC in the presence of oocyte extracts and various amounts of okadaic acid (ICN). Reactions were stopped by addition of 10 volumes of 20% TCA, centrifuged for 5 min, and the released [32P] was counted.

Results Immunoblotting Proteins were subjected to electrophoresis in Laemmli buffer on a 12% SDS-PAGE (Laemmli, 1970) and then transferred to nitrocellulose filters (Schleicher & Schull). Proteins of interest were visualized by using the appropriate primary antibody: rabbit anti-phosphoTyr15 Cdc2 and antiphosphoThr161 Cdc2 antibodies from Cell Signaling Technology, rabbit anti-PSTAIR antibody from Upstate Biotechnology, mouse antibody directed against the active phosphorylated form of mitogen-activated protein kinase (MAPK) and rabbit antibodies against the active phosphorylated form of MEK1/2 from New Englands Biolabs, goat anti-Rsk2 antibody and rabbit anti-ERK1/2 antibody from Santa Cruz Biotechnologies, mouse antibodies against the catalytic subunit of PP1 from Transduction Laboratories. The antibodies directed against Xenopus Cdc25 and the various subunits of Xenopus PP2A were previously described in Karaiskou et al. (1998). The primary antibodies were detected with appropriated horseradish peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch laboratories) and the Western blot Chemoluminescence Renaissance kit (Perkin Elmer Life Sciences). Histone H1 kinase assay Cdc2 activity (equivalent to five oocytes) was assayed using histone H1 (Boehringer) as a substrate after affinity purification on p13suc1-beads as described (Jessus et al., 1991). The histone H1 kinase activity was measured in a

Time-course of Cdc2 activation during meiotic maturation of axolotl oocyte Oocyte maturation was induced by 1 AM progesterone. As shown in Fig. 1, the time-course of oocyte maturation was followed and the morphological features of maturing oocytes were examined and called stages A – F, whereby stage A corresponds to unstimulated prophase oocytes. The first pigment rearrangement corresponded to the formation of a white and diffuse area formed at the animal pole (stage B). The translucent germinal vesicle appeared in the middle of the white area that became totally translucent when germinal vesicle migration was completed (stage C). Then, the translucent area was invaded by yolk-filled cytoplasm, corresponding to the full breakdown of the germinal vesicle, and became opaque (stage D). It has been shown in axolotl oocyte that as in Xenopus oocytes, breakdown of the nuclear envelope starts at the basal part of the germinal vesicle (Beetschen and Gautier, 1989). Therefore, since the beginning of GVBD is not externally visible, it is possible that it can be initiated in some of the stage C oocytes. Two hours after GVBD, a small bright area appeared in the center of the maturation spot, and was then encircled by a dark ring (stage E). According to previous reports (Beetschen and Gautier, 1989), this stage corresponds to the extrusion of the first polar body. The pigmented ring became smaller and darker (stage F), and this oocyte morphology was then stable for more than 16 h, corresponding to the metaphase II arrest observed in all vertebrate species.

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Fig. 1. (A) Six stages (A – F) of axolotl oocyte maturation. Stage A, prophase oocyte. Stage B, appearance of a white and diffuse area at the animal pole. Stage C, the translucent germinal vesicle is visible at the animal pole. Stage D, yolk-filled white area observed after completion of germinal vesicle breakdown. Stage E, extrusion of the first polar body surrounded by a pigmented ring. Stage F, metaphase II-arrested oocyte. Scale bar = 500 Am. (B) Prophase oocytes were incubated in the presence of progesterone, and percentage of GVBD was determined as a function of time by following the appearance of stage C – D.

Oocytes were selected at the different stages described in Fig. 1A and Cdc2 kinase activity was estimated. The use of the anti-PSTAIR antibody that recognizes specifically the PSTAIR motif conserved in Cdc2 from species ranging from yeast to man shows that Cdc2 is expressed in axolotl oocyte and that the overall level of the protein does not vary during the maturation process (Fig. 2A). In all species studied so far, Cdc2 exhibits a strong affinity for the yeast p13suc1 protein (Brizuela et al., 1989). Axolotl oocyte lysates were incubated in the presence of p13suc1-beads, and the proteins bound to the beads were analyzed by Western blot using the anti-PSTAIR antibody. Fig. 2A

shows that axolotl Cdc2 binds to p13suc1. To assay specifically Cdc2 activity during axolotl meiotic maturation, oocyte lysates were incubated in the presence of p13suc1beads, and histone H1 kinase activity was measured in the bead pellet. As shown in Fig. 2B, H1 kinase activity of Cdc2 is not detectable in prophase oocyte and starts to increase just before GVBD (stage C – D). Cdc2 kinase activity reaches a maximal level at the end of GVBD (Fig. 2B, stage D –E). At metaphase II (Fig. 2B, stage F), Cdc2 kinase activity declines slightly and reaches a lower level in metaphase II-arrested oocytes maintained for longer times (up to 24 h, data not shown) in culture. We did not attempt

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to detect the fluctuations in Cdc2 activity that are observed during the female meiotic interkinesis, since the requirement of five oocytes per each kinase assay probably introduces some level of asynchronicity. However, in some experiments, a decrease of Cdc2 activity was measured during the period of polar body extrusion (stage E), followed by a reincrease at stage F (metaphase II arrest). In Xenopus oocytes, the abrupt Cdc2 activation observed just before GVBD results from the conversion of the inactive pre-MPF molecules into active MPF, by Tyr15 dephosphorylation of cyclin B2-associated Cdc2. We investigated whether such a mechanism is also involved in Cdc2 activation in the axolotl oocyte. Tyr15 phosphorylation level of Cdc2 was analyzed by Western blot, using a monoclonal antibody recognizing specifically the Tyr15-phosphorylated residue of Cdc2 that is in a region strongly conserved in all the species studied until now. Fig. 2 shows that Tyr15phosphorylated Cdc2 is present in the prophase oocyte and that Cdc2 is dephosphorylated on Tyr15 before GVBD, in correlation with its kinase activation. In Xenopus oocytes, it is well established that MPF activation is negatively regulated by PKA activity (Maller and Krebs, 1980). Microinjection of the PKA inhibitor, PKI, in axolotl prophase oocytes induced GVBD and Cdc2 activation (data not shown), showing that PKA downregulation is sufficient to induce Cdc2 kinase activation in the axolotl oocyte. Inhibition of protein synthesis by cycloheximide totally prevented GVBD and Cdc2 activation induced by progesterone (data not shown), showing that under hormonal stimulation, MPF activation requires synthesis of new proteins in axolotl oocyte, as well as in Xenopus oocyte. MAP kinase is activated during meiotic maturation of axolotl oocytes but is not required for Cdc2 activation and GVBD

Fig. 2. Cdc2 and MAP kinase activation during axolotl oocyte meiotic maturation. (A) Extracts of prophase oocytes (Pro) and metaphase IIoocytes (MII) were incubated on p13suc1-beads. Total oocyte extracts (three oocytes per lane) and pull-down proteins bound to p13suc1-beads (20 oocytes per lane) were analyzed by Western blot with antibodies directed against the PSTAIR motif (Cdc2) and the anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2). (B) Prophase oocytes were incubated in the presence of progesterone and collected after progesterone addition at the different stages (A – F) defined in Fig. 1A. Lysates were subjected to H1 kinase assay (five oocytes per lane) or to Western blot (three oocytes per lane). From top to bottom: H1 kinase activity of Cdc2 assayed on p13 suc1 -beads; corresponding autoradiogram of radioactive histone H1; Western blots with the anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2); the anti-phospho MEK antibody (P-MEK); the anti-MAPK antibody (ERK1 and ERK2); the anti-phospho MAPK antibody (P-MAPK); and the anti-p90Rsk (Rsk) antibody. In this experiment (the same illustrated in Fig. 1B), GVBD occurred 5 h after progesterone addition.

MAP kinase (MAPK) is activated during the meiotic maturation process of all species studied so far and plays a crucial role in the control of the metaphase I– II transition and the meiotic arrest that occurs before fertilization (Dupre et al., 2002; Tachibana et al., 2000; Verlhac et al., 1996). However, its implication in the first Cdc2 activation wave that precedes GVBD represents a controversial issue (Dupre et al., 2002; Fisher et al., 1999; Gross et al., 2000). MAPK activation time-course In an attempt to get some insights in this matter, we investigated MAPK activation during meiotic maturation of axolotl oocyte. MAPK is expressed at the same protein level in prophase and metaphase II-arrested oocytes under two forms, presumably ERK1 and ERK2, as detected by Western blot using a polyclonal anti-ERK antibody (Fig. 2B). The electrophoretic mobility of both bands is partially retarded before GVBD, suggesting that MAPK was activated during meiotic maturation. MAPK activity was assessed

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during this process by using an anti-phospho MAPK antibody that recognizes specifically the active phosphorylated forms of ERK1 and ERK2. As shown in Fig. 2B, ERK1 and ERK2 are inactive in prophase oocyte, and both are activated before GVBD. MAPK is activated most probably by MEK1 and MEK2, both of which are activated concomitantly to ERK1/2, as judged by Western blot using an antiphospho MEK antibody that recognizes specifically the active phosphorylated forms of MEK1/2 (Fig. 2B). The first characterized substrate of MAPK in vertebrate oocyte, p90Rsk, is also present in axolotl oocyte, and its electrophoretically retarded form is observed concomitantly to MAPK

activation, before GVBD, suggesting that p90Rsk is activated during the maturation process by MAPK (Fig. 2B). Although we were not able to detect Mos by using several antibodies directed against Xenopus Mos, it appears that the MAPK cascade already characterized in starfish, Xenopus, and mouse oocytes is also functional in axolotl oocytes. The Mos/MAPK cascade is able to induce Cdc2 activation To understand the interconnections between MAPK and Cdc2 in the axolotl oocyte, mRNA encoding mouse Mos was injected into prophase oocytes, in the absence of progesterone. Seven hours later, GVBD occurred and

Fig. 3. MAP kinase activity is not required for Cdc2 activation in response to progesterone in axolotl oocyte. (A) Mouse Mos mRNA was injected into prophase oocytes. Oocytes were collected at time of injection (time 0), 7.5 or 24 h later. H1 kinase activity was assayed on p13suc1-beads and lysates were analyzed by Western blot with the anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2) or the anti-phospho MAP kinase antibody (P-MAPK). Autoradiogram of radioactive histone H1 is also illustrated. GVBD is indicated by (+) and absence of GVBD by ( ). (B) Prophase oocytes were incubated or not in the presence of 50 AM U0126 and then stimulated by progesterone 3 h later. The percentage of GVBD was determined as a function of time by following the appearance of stage C – D. (C) Prophase oocytes were incubated in the presence of 50 AM U0126, and progesterone was added 3 h after. Oocytes were collected at the time of progesterone addition (time 0), 9.5 or 24 h later. As controls, prophase and metaphase II oocytes were also analyzed. H1 kinase activity was assayed on p13suc1beads and lysates were analyzed by Western blot with the anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2) or the anti-phospho MAP kinase antibody (P-MAPK). Autoradiogram of radioactive histone H1 is also illustrated. GVBD is indicated by (+) and absence of GVBD by ( ).

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Cdc2 kinase was activated as judged by its H1 kinase activity, although Tyr15 dephosphorylation was not yet fully completed at this time (Fig. 3A). MAPK was stably activated upon Mos mRNA injection (Fig. 3A). Therefore, axolotl MAPK is able to be activated by Mos and to induce Cdc2 activation and GVBD. The Mos/MAPK cascade is not required for Cdc2 activation induced by progesterone We then addressed the question of whether MAPK is recruited by progesterone to lead to the activation of Cdc2.

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Oocytes were incubated in the presence of U0126, a pharmacological inhibitor of MEK (Favata et al., 1998), and were then stimulated by progesterone. GVBD was retarded in the presence of U0126, in comparison to control progesterone-treated oocytes (Fig. 3B), and the white spot was not well organized. In some females, GVBD was partly inhibited (Fig. 3B: 55% GVBD 24 h after progesterone stimulation). As expected, MAPK activation was totally prevented under these conditions, for at least 24 h (Fig. 3C). However, in matured oocytes, Cdc2 kinase was activated as judged by H1 kinase assay, and Cdc2 was partially

Fig. 4. Injection of Cdc25 or cytoplasm from matured oocyte induces Cdc2 activation in axolotl oocyte. (A) Prophase oocytes were injected with Cdc25 in the presence (+) or in the absence ( ) of cycloheximide (CHX). They were homogenized at the indicated times after injection and assayed for histone H1 kinase activity of Cdc2 or analyzed by Western blot with the anti-phospho MAPK antibody (P-MAPK). Autoradiogram of radioactive histone H1 is also illustrated. GVBD is indicated by (+) and absence of GVBD by ( ). (B) Prophase oocytes (R1) were injected with cytoplasm from progesterone-matured oocytes. At GVBD, cytoplasm of R1 oocytes was injected into prophase R2 oocytes. The experiment was performed in the presence (+) or in the absence ( ) of cycloheximide (CHX). R1 and R2 oocytes were homogenized at time of GVBD and assayed for histone H1 kinase activity of Cdc2. Autoradiogram of radioactive histone H1 is also illustrated. Pro: prophase control oocytes; Pg: oocytes at GVBD after progesterone treatment. The scheme depicts the experimental procedure.

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dephosphorylated on Tyr15, at GVBD (Fig. 3C). Therefore, as in Xenopus oocyte (Gross et al., 2000), MAPK activity is not necessary in axolotl oocytes for MPF activation induced by progesterone, although it contributes to this process. Presence of pre-MPF in immature prophase oocytes of axolotl It has been proposed that pre-MPF is absent in the immature oocyte of most species of fishes and amphibians,

except Xenopus, and that activation of MPF induced by steroids in these cells would rely on cyclin B synthesis (Tanaka and Yamashita, 1995). As shown in Figs. 2 and 3, a population of Cdc2 molecules is phosphorylated on Tyr15 residue in axolotl prophase oocyte. Since this inhibitory phosphorylation of Cdc2 takes place at the time of the association with its cyclin partner, these data strongly suggest that this inactive Tyr15-phosphorylated Cdc2 population is associated with cyclin, and therefore represents pre-MPF. None of the numerous anti-cyclin B antibodies

Fig. 5. Activation of Cdc2 by Cdc25 and okadaic acid in extracts from axolotl prophase oocyte. (A) Extracts from prophase oocytes were supplemented with an ATP-regenerating system and Cdc25 phosphatase and were incubated at 30jC. Samples were collected at various times for histone H1 kinase assay and analysis by Western blot with anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2) or the anti-phospho MAP kinase antibody (P-MAPK). Autoradiogram of radioactive histone H1 is also illustrated. (B) Axolotl oocyte prophase oocytes were injected with okadaic acid in the presence (+) or in the absence ( ) of cycloheximide (CHX). They were homogenized at the indicated times after injection and assayed for histone H1 kinase activity of Cdc2. Autoradiogram of radioactive histone H1 is also illustrated. (C) Extracts from prophase oocytes were supplemented with an ATP-regenerating system and okadaic acid and were then incubated at 30jC. Samples were collected at various times for histone H1 kinase assay. Autoradiogram of radioactive histone H1 is also illustrated.

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that we have tested were able to recognize any protein in both prophase and metaphase II axolotl oocytes. We therefore investigated the presence of pre-MPF by indirect approaches. In the absence of progesterone, we injected two agents known to activate pre-MPF molecules into prophase oocytes: Cdc25, the phosphatase responsible for the activating dephosphorylation of Thr14 and Tyr15 residues of Cdc2 (Jessus and Ozon, 1995), and cytoplasm originating from metaphase II-arrested oocytes, responsible for the auto-amplification mechanism that allows the abrupt conversion of pre-MPF into MPF (Masui and Markert, 1971). These activators of pre-MPF were injected in the presence of cycloheximide to prevent synthesis of proteins, especially cyclin. Both of them induced GVBD and Cdc2 activation within 1 – 3 h, independently of protein synthesis (Fig. 4). Moreover, oocytes induced to mature by injection of cytoplasm were able to develop transferable MPF activity, as injection of their cytoplasm induced GVBD in recipient oocytes, independently of protein synthesis (Fig. 4B). This demonstrates that Cdc2 can be directly activated in the absence of cyclin synthesis and that pre-MPF is present in the axolotl full-grown immature oocyte. Interestingly, Cdc25 injection and cytoplasm transfer were able to induce MAPK activation, in a protein synthesis-dependent manner (Fig. 4A and data not shown). This means that MAPK can be activated under the control of Cdc2, and that activation of this enzyme depends on the synthesis of a new protein, presumably a c-Mos ortholog. To ascertain the presence of pre-MPF, we developed an in vitro approach, based on the use of prophase oocyte extracts, as previously described in the Xenopus system (Karaiskou et al., 1998, 1999). Diluted axolotl prophase oocyte lysates were centrifuged at low speed. The supernatant was then complemented with an ATP-regenerating system, Cdc25, and incubated at 30jC. These extracts do not support any protein synthesis and allow the analysis of Cdc2 and MAPK activation induced by the direct activator of pre-MPF, Cdc25. Addition of Cdc25 in such extracts induced H1 kinase activation and Tyr15 dephosphorylation of Cdc2 within 1 h (Fig. 5A). As in Xenopus extracts (Karaiskou et al., 1999), MAPK was never activated under these conditions (Fig. 5A), indicating that a crucial protein is missing in these extracts to allow Cdc2 to activate MAPK, most probably Mos. This result reinforces the in vivo data, confirming that the prophase axolotl oocyte contains pre-MPF molecules that can be directly activated by Tyr15 dephosphorylation of Cdc2 in the absence of cyclin synthesis. MPF auto-amplification feedback loop is not functional in extracts from axolotl prophase oocytes In Xenopus prophase oocytes, it has been described that okadaic acid, a specific inhibitor of Ser/Thr protein phosphatase 2A that regulates Cdc25 activity, leads to MPF autoamplification (Felix et al., 1990; Goris et al., 1989; Kar-

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Fig. 6. Presence of an active phosphatase 2A sensitive to okadaic acid in axolotl prophase oocyte. (A) Prophase oocytes (Pro) and metaphase IIoocytes (MII) were homogenized and subjected to Western blots with antibodies directed against the catalytic subunit of PP1 (C36), the 65 and 55 kDa regulatory subunits of PP2A (respectively, R65 and R55), the catalytic subunit of PP2A (C35), the Cdc25 phosphatase, and the polo-like kinase. Migration position of molecular weight markers is indicated on the left. (B) 32 P-phosphorylated casein was incubated for 20 min at 30jC in the presence of various volumes of extracts from prophase or metaphase IIoocytes (upper panel), or in the presence of 10 Al of prophase extract and various concentrations of okadaic acid. The release of 32P from casein was expressed as % of control in the absence of extract and okadaic acid.

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aiskou et al., 1999). In a similar manner, injection of okadaic acid in axolotl prophase oocytes induced GVBD and Cdc2 kinase activation independently of protein synthesis (Fig. 5B). This result strengthens the conclusion that the MPF auto-amplification loop is functional in axolotl oocyte, as shown previously by Cdc25 injection and MPF transfer (Fig. 4). Interestingly, addition of okadaic acid in axolotl prophase extracts did not lead to Cdc2 kinase activation (Fig. 5C). Various concentrations of okadaic acid (from 10 10 to 10 5 M) were tested, as well as other PP1 and PP2A inhibitors (such as tautomycin), various centrifugation speed extracts (from 1000 to 100 000  g), various dilutions of the extracts, and several incubation temperatures, all these conditions being not sufficient to lead to Cdc2 activation. Addition of Cdc25 together with okadaic acid did not modify the time-course and the extent of Cdc2 activation normally induced by Cdc25 (data not shown). However, as in Xenopus oocyte extracts, MAPK was activated in response to okadaic acid, indicating that an okadaic acid-sensitive phosphatase is present in the extracts (data not shown). To ascertain the presence of the targets of okadaic acid in the extracts, Western blots using anti-PP1 and PP2A antibodies were performed. The catalytic subunits of both phosphatases are expressed in the axolotl oocyte, as well as the 55 and 65 kDa regulatory subunits of PP2A (Fig. 6A). We assayed PP2A activity in the extracts by using as a

substrate PKA-phosphorylated casein, a specific in vitro substrate of this phosphatase. Fig. 6B shows that PP2A is active and inhibited by nanomolar concentrations of okadaic acid in prophase or metaphase II oocyte extracts. Therefore, the inability of okadaic acid to induce Cdc2 activation in axolotl oocyte extracts does not result from a lack of PP2A, but rather resides at the level of other regulators of Cdc2 auto-amplification, absent or not accessible in the extracts but available in the intact cell. It has been demonstrated in the Xenopus system that Cdc25 and Plx1 are essential elements of MPF auto-amplification, the first one being responsible for Cdc2 dephosphorylation, the second one regulating Cdc25 (Abrieu et al., 1998; Karaiskou et al., 1999; Kumagai and Dunphy, 1996). We ascertained the presence of both Cdc25 and Plx1 in axolotl oocyte by Western blotting, using anti-Cdc25 and anti-polo polyclonal antibodies (Fig. 6A). Both proteins are expressed at a similar level in prophase and metaphase IIoocytes and undergo a retardation of their electrophoretic mobility during meiotic maturation, characteristic of their activation (Fig. 6A), as shown in Xenopus. Therefore, preMPF and the known regulators of the MPF positive feedback loop are expressed in axolotl prophase oocyte. Although MPF auto-amplification can be induced in vivo by okadaic acid injection, this inhibitor is unable to trigger this process in vitro, indicating the important role of the intracellular compartmentalization in ovo.

Fig. 7. Injection of cyclin A induces Cdc2 activation in axolotl oocyte. Prophase oocytes were injected with cyclin A in the presence (+) or in the absence ( ) of cycloheximide (CHX). They were homogenized at the indicated times after injection and assayed for histone H1 kinase activity of Cdc2 (autoradiogram of radioactive histone H1 is also illustrated) or analyzed by Western blot with the anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2) or the anti-phospho MAPK antibody (P-MAPK). GVBD is indicated by (+) and absence of GVBD by ( ).

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pre-MPF represents a minor fraction of Cdc2 molecules in immature prophase oocyte of axolotl In prophase oocyte of all species studied until now, monomeric molecules of Cdc2 are present in excess compared to Cdc2 molecules involved in pre-MPF (De Smedt et al., 2002; Kobayashi et al., 1991). To investigate whether such a pool of monomeric Cdc2 molecules is present in axolotl oocyte, cyclin was injected into prophase oocytes, in the presence or in the absence of cycloheximide. For these experiments, we used recombinant human cyclin A, known as a potent activator of Cdc2 in Xenopus oocyte (Clarke et al., 1992; Lorca et al., 1992). In both cases, injection of cyclin A led to GVBD, to Cdc2 kinase activation, and to Tyr15 dephosphorylation of Cdc2 (Fig. 7). MAPK was also activated in a protein synthesis-dependent manner (Fig. 7). These results indicate that injected cyclin A is able to recruit and activate endogenous monomeric Cdc2 molecules that in turn induce the activation of pre-MPF stored in the prophase oocyte. We undertook a quantitative analysis to estimate the ratio of free Cdc2 molecules versus pre-MPF. Axolotl prophase and metaphase II-oocyte extracts were fractionated by gel filtration, and fractions were analyzed by Western blotting with three antibodies: the anti-PSTAIR antibody reveals all forms of Cdc2; the anti-phosphoTyr Cdc2 antibody reveals inactive Cdc2 molecules bound to cyclin; the anti-phosphoThr161 Cdc2 antibody reveals Cdc2 molecules phosphorylated on Thr161, a phosphorylation required for Cdc2 activity. As already described in Xenopus (De Smedt et al., 2002), the anti-PSTAIR antibody revealed the presence of Cdc2 in fractions 7 and 9 (Fig. 8A). Fractions 7 and 9 correspond to proteins of molecular weights around 100 –70 and 40– 20 kDa, respectively, suggesting that Cdc2 is under a monomeric form in fraction 9, and bound to proteins of 40 –60 kDa in fraction 7. To ascertain that Cdc2 present in fraction 9 is under a monomeric form, its potential for activation by cyclin was analyzed. Fraction 9 was supplemented with cyclin A, in the presence or in the absence of Civ1, the S. cerevisiae protein kinase responsible for the activating Thr phosphorylation of the yeast cyclin-dependent kinase (CDK) T-loop (Espinoza et al., 1996; Kaldis et al., 1996; Thuret et al., 1996). Cyclin A addition gave raise to a high Cdc2 kinase activity in the presence of Civ1, as well as the phosphorylation of Cdc2 on Thr161, whereas Cdc2 was not activated by cyclin in the absence of the yeast kinase (Fig. 8B). This result demonstrates that fraction 9 contains monomeric Cdc2 molecules that are not phosphorylated on Thr161, but that are available to cyclin binding and activation in a Thr161-phosphorylation-dependent manner. In contrast, addition of cyclin A and Civ1 to fraction 7 did not generate any Cdc2 kinase activation (Fig. 8B). Moreover, Cdc2 detected in fraction 7 from prophase oocytes is phosphorylated on Tyr15 and Thr161, whereas it is only phosphorylated on Thr161 when it originates from

Fig. 8. Separation of monomeric Cdc2 from pre-MPF complexes by gel filtration. (A) A cytosolic extract from prophase (Pro) or metaphase II (MII) axolotl oocytes was fractionated by Superose 12 chromatography. Fractions 7 (F7) and 9 (F9) were analyzed by Western blotting, using from top to lower panels: the anti-PSTAIR antibody (Cdc2), the anti-phosphoThr161 Cdc2 antibody (P-Thr Cdc2), and the anti-phosphoTyr Cdc2 antibody (PTyr Cdc2). (B) Fractions 7 (F7) and 9 (F9) from prophase oocytes were incubated at 30jC for 1 h in the presence or in the absence of cyclin A and/ or Civ1. Histone H1 kinase activity of Cdc2 was then assayed. Autoradiogram of radioactive histone H1 is illustrated. Western blots were performed with the anti-phosphoThr161 Cdc2 antibody (P-Thr Cdc2) and the anti-phosphoTyr Cdc2 antibody (P-Tyr Cdc2).

metaphase oocytes (Fig. 8). These observations, together with the expected molecular weight of complexes recovered in this fraction, show that Cdc2 present in fraction 7 is bound to cyclin and represents pre-MPF. Interestingly, Cdc2 is much more abundant in fraction 9 than in fraction 7, in a similar manner in prophase and matured-oocytes, showing that monomeric Cdc2 molecules of fraction 9 are much more abundant than bound Cdc2 of fraction 7 (Fig. 8A).

Discussion Although essentially everything known about the mechanisms of MPF activation in oocyte has come from studies with X. laevis (Karaiskou et al., 2001; Nebreda and Ferby, 2000), it has been proposed that several species of fishes

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and amphibians could employ different mechanisms of MPF activation during oocyte maturation (Hirai et al., 1992; Katsu et al., 1993; Tanaka and Yamashita, 1995; Yamashita et al., 1995). Two main types of mechanisms of MPF activation during oocyte meiotic maturation would be operating. It is well established that in X. laevis, pre-MPF, consisting of cyclin B-bound Cdc2 phosphorylated on Thr14 and Tyr15, is stockpiled in full-grown prophase oocytes (Kobayashi et al., 1991). Dephosphorylation of Cdc2 on Thr14 and Tyr15 is primarily responsible for MPF activation through the auto-amplification mechanism, and the synthesis of cyclin B is not necessary for entry into meiosis I (Hochegger et al., 2001). In this case, it is possible to induce MPF activation in the absence of protein synthesis by injecting into Xenopus oocyte either the Cdc25 phosphatase, or okadaic acid, a PP2A inhibitor, or cytoplasm taken from metaphase II-arrested oocytes. These three factors are able to act at the level of pre-MPF and to trigger its conversion into MPF independently of protein synthesis (Goris et al., 1989; Rime et al., 1994; Wasserman and Masui, 1975). MPF activation in the oocyte of another Xenopus species, X. tropicalis, successfully introduced for genetic studies of development, appears very similar to what is known in X. laevis (Bodart et al., 2002; Stanford et al., 2003). In striking contrast, pre-MPF is absent in immature oocytes of goldfish, carp, catfish, lamprey, Rana, and Bufo. In full-grown prophase oocytes of these species, cyclin B is not expressed at the protein level and all Cdc2 exists under monomeric form (Kajiura et al., 1993; Tanaka and Yamashita, 1995). This implies that de novo synthesis of cyclin B, its binding to Cdc2, and the subsequent activating Thr161 phosphorylation of Cdc2 are critical steps for MPF activation. These observations raise the question of whether much of what we have learned from studies of X. laevis oocytes holds for those of other animal species. For comprehensive understanding of the molecular mechanisms of MPF activation, as well as oocyte maturation, we carried out the present study using the axolotl urodele amphibian. The first point established here demonstrates that the transduction pathway induced by the steroid maturation trigger, progesterone, exhibits strong similarities in axolotl oocyte with the pathway operating in Xenopus oocyte. First, Cdc2 is activated at the same time than MAPK, just before GVBD. Second, although Mos kinase is able to activate MAPK and to trigger Cdc2 activation and entry into meiosis I in the absence of progesterone, MAPK is not required for MPF activation induced by the hormone. However, the delay in GVBD observed in the absence of MAPK activation suggests that the MAPK pathway contributes to Cdc2 activation. Third, repressing PKA activity induces GVBD in the absence of progesterone, and MPF activation induced by the hormone depends on protein synthesis. Thus, the signaling pathways regulating oocyte maturation in Xenopus and axolotl appear to be extremely similar. The main difference resides at the level of the lag period preceding MPF activation, longer in axolotl (7 h) than in X. laevis (3–

4 h). This could reflect a difference in the stage at which these prophase oocytes are arrested. As already noticed (Yamashita et al., 1995), oocytes that have the germinal vesicle located deeply in the cytoplasm and migrating toward the animal pole in response to the steroid inducer, such as goldfish (Yamamoto and Yamazaki, 1961) or axolotl oocytes (this study), could be arrested at a stage more distal to GVBD than oocytes where the germinal vesicle is near the animal pole as in Xenopus. The second point established by our work is the presence of pre-MPF in the axolotl immature full-grown oocyte. We were not able to directly demonstrate the presence of cyclin B in the axolotl prophase oocyte, given the annoying observation that all antibodies against cyclin B tested did not show any cross-reactivity with axolotl proteins. However, the presence of pre-MPF is firmly established by the following data. First, Cdc2 is phosphorylated on Tyr15 in prophase oocyte, a phosphorylation state characterizing cyclin B-bound Cdc2 and not monomeric Cdc2 (Norbury et al., 1991). Second, Tyr15-phosphorylated Cdc2 is recovered by gel filtration in a fraction corresponding to proteins of molecular weights around 100 – 70 kDa, suggesting that Cdc2 is bound to proteins of 40 –60 kDa, most probably cyclins. Third, Cdc2 can be activated in vitro in diluted oocyte extracts by addition of Cdc25 phosphatase, independently of protein synthesis. Fourth, MPF activation and GVBD are induced in vivo in the absence of protein synthesis by microinjection of three factors well known to trigger the conversion of pre-MPF into MPF: either Cdc25, or okadaic acid, or cytoplasm transfer. Therefore, in contrast to many fishes and amphibians oocytes (except Xenopus), the axolotl immature oocyte has a stockpile of inactive preMPF, containing Tyr15-phosphorylated Cdc2 that can be activated directly by the Cdc25 phosphatase. Despite the presence of pre-MPF, three significant differences were observed between Xenopus and axolotl MPF activation. First, Tyr15 dephosphorylation and activation of axolotl Cdc2 are not abrupt events taking place within a very short period as in Xenopus oocyte. It is remarkable that at GVBD, Cdc2 is not yet fully dephosphorylated on Tyr15 despite the presence of active Cdc2 molecules (see Figs. 2, 3, and 7). An inactive Tyr15-phosphorylated population of Cdc2 molecules coexists with active Cdc2 kinase in the cell before the complete Tyr15 dephosphorylation of Cdc2 is progressively reached. It is established in Xenopus oocyte that the first molecules of active Cdc2 contribute to the activating phosphorylations of the Cdc25 phosphatase: the more Cdc2 is activated, the more it activates Cdc25, in turn activating Cdc2, resulting in a strong acceleration and the abruptness of MPF activation (Hoffmann et al., 1993; Karaiskou et al., 1999; Masui and Markert, 1971). In axolotl oocytes, the coexistence of active MPF with Tyr15-phosphorylated Cdc2 molecules for about 1 h at the time of GVBD and the slow and progressive Tyr15-dephosphorylation of pre-MPF indicates that the interaction between Cdc2 and Cdc25 operates in a different way in axolotl and

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Xenopus oocytes. Although the ability of a small amount of active MPF to trigger the conversion of pre-MPF into MPF is working in the axolotl oocyte, as cytoplasm transfer induces Cdc2 activation and GVBD in the absence of protein synthesis in a transferable manner, it does not allow a rapid self-amplification of MPF as in Xenopus oocyte. The large size of the axolotl oocyte could result in an asynchronous activation of MPF, accounting for this phenomenon. The second noticeable point is that the MPF auto-amplification mechanism does not take place in axolotl oocyte extract. We have previously described the effects of okadaic acid addition in a cell-free system derived from Xenopus prophase oocytes: abrupt onset of the MPF auto-amplification loop, including Cdc25 hyper-phosphorylation and Cdc2 activation, an event requiring ATP and Plx1 kinase activity (Karaiskou et al., 1998, 1999). Interestingly, although injection of okadaic acid in axolotl prophase oocyte induces GVBD and MPF activation, and although all the known components of the MPF feedback loop are present in the extract (Cdc2, Cdc25, Plx1, and PP2A), this inhibitor has no effect when added to axolotl oocyte extract, regardless of the extract dilution, the centrifugation speed, the incubation temperature or time. Therefore, the MPF auto-amplification mechanism probably depends on a precise organization of intracellular compartments that is disrupted in oocyte extracts. This regulation level that is not strongly exerted in the Xenopus oocyte could account for the in vivo characteristics of the axolotl MPF activation mechanism. A third original point of the axolotl oocyte is the very high ratio of monomeric Cdc2 versus Tyr15-phosphorylated Cdc2. Monomeric Cdc2 is present in excess over cyclin Bbound Cdc2 in oocytes of all species studied so far (80% of the total population of Cdc2 in the Xenopus oocyte; Kobayashi et al., 1991). In axolotl, we have shown by gel filtration analysis that pre-MPF is almost negligible compared to the huge population of monomeric Cdc2 that is directly accessible to cyclin binding and activation, depending on concomitant Thr161 phosphorylation. Therefore, the low level of preMPF could also be limiting in the function of the feedback loop operating in the axolotl oocyte. MPF activation in the axolotl oocyte could represent an intermediate category between the two extreme models described until now. As the Xenopus oocyte, the axolotl oocyte has a pre-MPF store. Theoretically, MPF activation could take place abruptly, independently of cyclin B synthesis, based on self-amplification mechanism. This is not the case, mainly due to the very low level of pre-MPF and the progressive and slow Tyr15dephosphorylation of Cdc2. It is therefore possible that as it is the case in goldfish and several anoura species, cyclin B synthesis contributes to MPF activation in axolotl oocyte.

Acknowledgments We thank all members of our laboratories, especially Pr. Rene´ Ozon, Dr. Olivier Haccard, and Dr. Xavier Cayla for

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helpful discussions, advises, and technical assistance. We are grateful to Dr. J. Maller (HHMI, USA) for the antiCdc25 antibody, to Dr. C. Mann (CEA, France) for yeast recombinant Civ1 protein, and to Dr. M.H. Verlhac (CNRS, France) for mouse Mos mRNA. This research was supported by grants from INRA, CNRS, University Paris VI, ARC (No. 4771 to C.J.) and Ligue Nationale contre le Cancer (to Y.A. and C.J.). References Abrieu, A., Brassac, T., Galas, S., Fisher, D., Labbe, J.C., Doree, M., 1998. The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs. J. Cell Sci. 111, 1751 – 1757. Beetschen, J.C., Gautier, J., 1989. In: Armstrong abd, J.B., Malacinski, G.M. (Eds.), Developmental Biology of the Axolotl. Oxford Univ. Press, New York, Oxford, pp. 25 – 35. Bodart, J.F., Gutierrez, D.V., Nebreda, A.R., Buckner, B.D., Resau, J.R., Duesbery, N.S., 2002. Characterization of MPF and MAPK activities during meiotic maturation of Xenopus tropicalis oocytes. Dev. Biol. 245, 348 – 361. Brizuela, L., Draetta, G., Beach, D., 1989. Activation of human Cdc2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit. Proc. Natl. Acad. Sci. U. S. A. 86, 4362 – 4366. Clarke, P.R., Leiss, D., Pagano, M., Karsenti, E., 1992. Cyclin A-dependent and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts. EMBO J. 11, 1751 – 1761. Coleman, T.R., Dunphy, W.G., 1994. Cdc2 regulatory factors. Curr. Opin. Cell Biol. 6, 877 – 882. De Smedt, V., Poulhe, R., Cayla, X., Dessauge, F., Karaiskou, A., Jessus, C., Ozon, R., 2002. Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes. J. Biol. Chem. 277, 28592 – 28600. Dunphy, W.G., Kumagai, A., 1991. The Cdc25 protein contains an intrinsic phosphatase activity. Cell 67, 189 – 196. Dunphy, W.G., Brizuela, L., Beach, D., Newport, J., 1988. The Xenopus Cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54, 423 – 431. Dupre, A., Jessus, C., Ozon, R., Haccard, O., 2002. Mos is not required for the initiation of meiotic maturation in Xenopus oocytes. EMBO J. 21, 4026 – 4036. Espinoza, F.H., Farrell, A., Erdjument-Bromage, H., Tempst, P., Morgan, D.O., 1996. A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK. Science 273, 1714 – 1717. Favata, M.F., Horiuchi, K.Y., Manos, E.J., Daulerio, A.J., Stradley, D.A., Feeser, W.S., Van Dyk, D.E., Pitts, W.J., Earl, R.A., Hobbs, F., Copeland, R.A., Magolda, R.L., Scherle, P.A., Trzaskos, J.M., 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623 – 18632. Felix, M.A., Cohen, P., Karsenti, E., 1990. Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid. EMBO J. 9, 668 – 675. Fisher, D.L., Brassac, T., Galas, S., Doree, M., 1999. Dissociation of MAP kinase activation and MPF activation in hormone-stimulated maturation of Xenopus oocytes. Development 126, 4537 – 4546. Frank-Vaillant, M., Jessus, C., Ozon, R., Maller, J.L., Haccard, O., 1999. Two distinct mechanisms control the accumulation of Cyclin B1 and Mos in Xenopus oocytes in response to progesterone. Mol. Biol. Cell 10, 3279 – 3288. Gautier, J., Norbury, C., Lohka, M., Maller, J., 1988. Purified maturation promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle gene cdc2+. Cell 54, 433 – 439. Gautier, J., Matsukawa, T., Nurse, P., Maller, J., 1989. Dephosphorylation

278

S. Vaur et al. / Developmental Biology 267 (2004) 265–278

and activation of Xenopus p34cdc2 protein kinase during the cell cycle. Nature 339, 626 – 629. Gautier, J., Solomon, M.J., Booher, R.N., Bazan, J.F., Kirschner, M.W., 1991. Cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell 67, 197 – 211. Goris, J., Hermann, J., Hendrix, P., Ozon, R., Merlevede, W., 1989. Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett. 245, 91 – 94. Gross, S.D., Schwab, M.S., Taieb, F.E., Lewellyn, A.L., Qian, Y.W., Maller, J.L., 2000. The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk). Curr. Biol. 10, 430 – 438. Gurdon, J.B., Wickens, M.P., 1983. The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 101, 370 – 386. Hirai, T., Yamashita, M., Yoshikuni, M., Lou, Y.H., Nagahama, Y., 1992. Cyclin-B in fish oocytes—Its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2-activation. Mol. Reprod. Dev. 33, 131 – 140. Hochegger, H., Klotzbucher, A., Kirk, J., Howell, M., le Guellec, K., Fletcher, K., Duncan, T., Sohail, M., Hunt, T., 2001. New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation. Development 128, 3795 – 3807. Hoffmann, I., Clarke, P.R., Marcote, M.J., Karsenti, E., Draetta, G., 1993. Phosphorylation and activation of human Cdc25-C by Cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J. 12, 53 – 63. Jessus, C., Ozon, R., 1995. Function and regulation of Cdc25 phosphatase through mitosis and meiosis. Prog. Cell Cycle Res. 1, 215 – 228. Jessus, C., Rime, H., Haccard, O., Van, L.J., Goris, J., Merlevede, W., Ozon, R., 1991. Tyrosine phosphorylation of p34cdc2 and p42 during meiotic maturation of Xenopus oocyte. Antagonistic action of okadaic acid and 6-DMAP. Development 111, 813 – 820. Kajiura, H., Yamashita, M., Katsu, Y., Nagahama, Y., 1993. Isolation and characterization of goldfish Cdc2, a catalytic component of maturationpromoting factor. Dev. Growth Differ. 35, 647 – 654. Kaldis, P., Sutton, A., Solomon, M.J., 1996. The Cdk-activating kinase (CAK) from budding yeast. Cell 86, 553 – 564. Karaiskou, A., Cayla, X., Haccard, O., Jessus, C., Ozon, R., 1998. MPF amplification in Xenopus oocyte extracts depends on a two-step activation of Cdc25 phosphatase. Exp. Cell Res. 244, 491 – 500. Karaiskou, A., Jessus, C., Brassac, T., Ozon, R., 1999. Phosphatase 2A and polo kinase, two antagonistic regulators of Cdc25 activation and MPF auto-amplification. J. Cell Sci. 112, 3747 – 3756. Karaiskou, A., Dupre, A., Haccard, O., Jessus, C., 2001. From progesterone to active Cdc2 in Xenopus oocytes: a puzzling signalling pathway. Biol. Cell 93, 35 – 46. Katsu, Y., Yamashita, M., Kajiura, H., Nagahama, Y., 1993. Behavior of the components of maturation-promoting factor, Cdc2 kinase and cyclin-B, during oocyte maturation of goldfish. Dev. Biol. 160, 99 – 107. Kobayashi, H., Minshull, J., Ford, C., Golsteyn, R., Poon, R., Hunt, T., 1991. On the synthesis and destruction of A- and B-type cyclins during

oogenesis and meiotic maturation in Xenopus laevis. J. Cell Biol. 114, 755 – 765. Kumagai, A., Dunphy, W.G., 1996. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 273, 1377 – 1380. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lorca, T., Devault, A., Colas, P., Van, L.A., Fesquet, D., Lazaro, J.B., Doree, M., 1992. Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts. FEBS Lett. 306, 90 – 93. Maller, J.L., Krebs, E.G., 1980. Regulation of oocyte maturation. Curr. Top. Cell Regul. 16, 271 – 311. Masui, Y., Markert, C.L., 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129 – 146. Nebreda, A.R., Ferby, I., 2000. Regulation of the meiotic cell cycle in oocytes. Curr. Opin. Cell Biol. 12, 666 – 675. Norbury, C., Blow, J., Nurse, P., 1991. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates. EMBO J. 10, 3321 – 3329. Nurse, P., 1990. Universal control mechanism regulating the onset of Mphase. Nature 344, 503 – 508. Rime, H., Huchon, D., De Smedt, V., Thibier, C., Galaktionov, K., Jessus, C., Ozon, R., 1994. Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I. Biol. Cell 82, 11 – 22. Schuetz, A.W., Samson, D., 1979. Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes. Dev. Biol. 68, 636 – 642. Stanford, J.S., Lieberman, S.L., Wong, V.L., Ruderman, J.V., 2003. Regulation of the G2/M transition in oocytes of Xenopus tropicalis. Dev. Biol. 260, 438 – 448. Tachibana, K., Tanaka, D., Isobe, T., Kishimoto, T., 2000. c-Mos forces the mitotic cell cycle to undergo meiosis II to produce haploid gametes. Proc. Natl. Acad. Sci. U. S. A. 97, 14301 – 14306. Tanaka, T., Yamashita, M., 1995. Pre-MPF is absent in immature oocytes of fishes and amphibians except Xenopus. Dev. Growth Differ. 37, 387 – 393. Thuret, J.Y., Valay, J.G., Faye, G., Mann, C., 1996. Civ1 (CAK in vivo), a novel Cdk-activating kinase. Cell 86, 565 – 576. Verlhac, M., Kubiak, J., Weber, M., Geraud, G., Colledge, W., Evans, M., Maro, B., 1996. Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development 122, 815 – 822. Wasserman, W.J., Masui, Y., 1975. Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes. Exp. Cell Res. 91, 381 – 388. Yamamoto, K., Yamazaki, F., 1961. Rhythm of development in the oocyte of the goldfish, Carassius auratus. Bull. Fac. Fish., Hokaido Univ. 12, 93 – 110. Yamashita, M., Kajiura, H., Tanaka, T., Onoe, S., Nagahama, Y., 1995. Molecular mechanisms of the activation of maturation-promoting factor during goldfish oocyte maturation. Dev. Biol. 168, 62 – 75.