Transient reactivation of CSF in parthenogenetic one-cell mouse embryos

Biology oj the Cell 91 (1999) 641-647 o 1999 Editions scientifiques et mCdicales Elsevier SAS. All rights reserved

641

Original article

Transient reactivation of CSF in parthenogenetic one-celi mouse embryos Maria A Ciemerych a *, Jacek Z Kubiak b * * aDepartment

of Embryology,

Institute of Zoology, Warsaw University, Krakowskie Przedmiescie26/‘28,

00-927 Warsaw, Poland; bBiologie Cellulaire du Eveloppement, UMR 7622-CNRS, Universitk Paris 6, BBtiment C-30, Case 53, 9, quai St-Bernard, 75005 Paris, France

During meiosis, the cytostatic factor (CSF) activity stabilizes the activity of the M-phase promoting factor (MPF) in metaphase II arrested vertebrate oocytes. Upon oocyte activation, the inactivation of both MPF and CSF enables the entry into the first embryonic mitotic cell cycle. Using a biological assay based on cell-fusion (hybrid between a parthenogenetically activated egg entering the first mitotic division and an activated oocyte), we observed that in activated mouse oocytes a first drop in CSF activity is detectable as early as 20 min post-activation. This suggests that CSF is inactivated upon MPF inactivation. However, CSF activity increases again to reach a maximum 60 min postactivation and gradually disappears during the following 40 min. Thus, in activated mouse oocytes (undergoing the transition to interphase) CSF activity fluctuates before definitive inactivation. We found that hybrids arrested in M-phase, thus containing CSF activity after oocyte activation, have activated forms of MAP kinases while hybrids in interphase have inactive forms of these enzymes. We postulate that CSF inactivation in mouse oocytes proceeds in two steps. The initial inactivation of CSF, required for MPF inactivation, is transient and does not require MAP kinase inactivation. The final inactivation of CSF, required for normal embryonic cell cycle progression, is dependent upon the inactivation of MAP kinases. 0 1999 editions scientifiques et mkdicales Elsevier SAS

cell cycle / MAP kinase / meiosis / mitosis / protein phosphorylation

INTRODUCTION The metaphase arrest in mouse oocytes is maintained by an activity called cytostatic factor (CSF) (Masui and Markert, 1971). CSF activity was first demonstrated in amphibian oocytes (Masui and Markert, 1971) by transferring cytoplasm from metaphase II arrested oocytes into one blastomere of a two-cell embryo. The injected blastomere was arrested in M-phase while the non-injected blasto-

Present addresses: * Dana Farber Cancer Institute, Harvard Medical School, Department of Cancer Biology, 44 Binney Street, Boston, MA 02115, USA. ** Correspondence and reprints: UPR 41 CNRS, Biologie et G&+tique du Dkveloppement LJniversit6 de Rennes 1, Facult6 de Mkdecine, 2, avenue Prof. Leon-Bernard, CS 34317, 35043 Rennes cedex, Fmnce

CSF after mouse oocyte activation

mere continued to cleave. An equivalent activity was also detected in mouse M II-arrested oocytes using the cell fusion technique (Kubiak et al, 1993). CSF was defined as an activity which stabilizes the M-phase promoting factor (MPF) (Masui and Markert, 1971). The first molecule implicated in the CSF activity was the protooncogene c-mos gene product (Sagata et al, 1989). Mos is the MAP kinase kinase kinase which activates the MAP kinase pathway during meiotic maturation (Posada et al, 1993; Nebreda and Hunt, 1993). MAP kinase also has a CSF activity (Haccard et al, 1993). Activation of MAP kinase involves its phosphorylation on threonine and tyrosine residues (Posada and Cooper, 1992). In mouse oocytes, activation of the MAP kinase pathway correlates with a shift in the electrophoretic mobility of the two isoforms of MAP

Ciemerych and Kubiak

642

kinase, ERK 1 and ERK 2 (for extracellular regulated kinase 1 and 2; Verlhac et al, 1993, 1994). Activation of MAP kinases in mouse oocytes takes place after activation of MPF and germinal vesicle breakdown (GVBD) (Verlhac et al, 1993). It seems that CSF develops already during the first meiotic M phase, and is transiently inactivated at anaphase I (Ciemerych and Kubiak, 1998). After activation of M II oocytes, MAP kinase activity drops progressively during 34 h post-activation (Verlhac et aI, 1994). Mos is also destroyed after MPF inactivation when MAP kinase activity drops (Weber et al, 1991). MPF is an heterodimer composed of a catalytic subunit, the p34 cdc2 kinase, homologue of the fission yeast cdc2 gene product, and a regulatory subunit, cyclin B (Lee and Nurse, 1987; Murray et al, 1989). Since inactivation of MPF requires the proteolytic degradation of cyclin B, CSF may act by preventing cyclin B degradation. In mouse oocytes, both cyclin B degradation and synthesis were detected during the M II arrest (Kubiak et al, 1993) suggesting that CSF inhibits partially the cyclin B degradation machinery rather than totally blocking it. The inactivation of CSF upon oocyte activation would enable the rapid proteolysis of cyclin B and thus MPF inactivation (Kubiak et al, 1993; Winston, 1997). However, CSF inactivation upon oocyte activation has never been observed directly and was only anticipated from the behavior of MPF and the activity of the cyclin B degradation machinery. Fertilization or parthenogenetic activation of oocytes triggers a transient increase in the cytoplasmic free calcium (Miyazaki, 1988) and leads to the release of the meiotic arrest by inactivation of MPF (Lorca ei ~2, 1993). It is known that CSF is inactivated by a calcium transient (Watanabe et a2, 1989). Thus, the calcium transients appear to play a key role in both CSF and MPF inactivation, leading to the exit from the metaphase arrest. However, the detailed analysis of the dynamics of MPF and CSF inactivation upon Xenopus oocyte activation has led to an apparent paradox: CSF activity was still detectable after MPF inactivation (Watanabe et al, 1991). These authors suggested that CSF inactivation follows the inactivation of MPF. However, such a sequence of events appears to contradict the proposed major role of CSF in stabilization of MPF. Degradation of Mos and inactivation of MAP kinases and their substrates (eg p90r~k)follow inactivation of MPF in mouse oocytes (Weber et al, 1991; Kubiak et al, 1993; Verlhac et aZ, 1994; Kalab et al, 1996). It seems therefore possible that in the mouse, as in Xenopus, the inactivation of these kinases provokes a drop in CSF activity. However, there are no studies showing the profile of CSF inactivation in mammalian oocyte. CSFafter mouseoocyte activation

Biology of the Cell 91 (19991 641-647

In this study we investigate the dynamics of CSF activity after mouse oocyte activation using a biological assay based on cell fusion method. We show that CSF activity fluctuates after oocyte activation. Inactivation of CSF proceeds in two steps: first, CSF is transiently downregulated by a mechanism independent from Mos degradation and MAP kinase inactivation to allow exit from the M II arrest. Second, the disappearance of CSF activity after the transition to the first embryonic cell cycle requires inactivation of the MAP kinase pathway.

MATERIALS AND METHODS GV stage oocytes Germinal vesicle stage, incompetent oocytes were recovered from ovaries of 12-14-day-old Fl (C57BllO x CBA/H) females. The oocytes surrounded by cumulus cells were released from ovarian follicles. Cumulus cells were removed by pipetting and oocytes were collected in M2 medium.

Ovulated oocytes Fl (C57BllO x CBA/H) females were induced to ovulate by intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG, Intervet) followed 48 h later by 10 IU of human chorionic gonadotrophin (hCG, Intervet). M II oocytes were recovered 17 h after injection of hCG. The cumulus cells were dispersed by hyaluronidase (150 IU/mL). Oocytes were stored in M2 medium before further manipulation.

Parthenogenetic activation of ovulated oocytes M II oocytes were activated by a 6-min incubation in 8% ethanol in M2 medium (Cuthbertson, 1983) and cultured in MEM medium at 37°C under 5% CO? in air.

Fusion procedure Zonae pellucidae of all oocytes and one-cell embryos were digested with pronase (0.5% in Ringer solution, Sigma). Parthenogenetically activated eggs entering the first mitotic division were fused with oocytes activated 15-120 min before fusion procedure. We also produced control hybrids between: MII-arrested oocytes with one-cell parthenogenetic embryos which started first mitotic division, two parthenogenetically activated oocytes entering mitosis or two oocytes activated 15-120 min before fusion procedure. In all experiments cells were agglutinated with each other using phytohaemagglutinin (150 mg/mL of BSA-free M2 medium, Sigma). Agglutinted pairs were exposed for 90 s to 45% polyethylene glycol (PEG 2000, Fluka) dissolved in BSA-free M2 medium as described previously by Czolowska et al (1986). PEG-treated pairs were cultured in M2 medium.

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Biology of the Cell 91 (1999) 641-647

lmmunofluorescence Hybrids were fixed and processed for immunofluorescence as previously described (Maro et al, 1984modified by de Pennart et al, 1988). For tubulin staining, the rat monoclonal antibody YL1/2 was used (Kilmartin et al, 1982) followed by fluorescein labeled anti-rat antibody (Biosys). Chromatin was visualized with propidium iodide. The samples were mounted in ‘Citifluor’ and observed using a Bio-Rad MRC-600 laser scanningconfocal microscope.

lmmunoblotting Five hybrid cellswere washed in M2 + PVP and then collected in 5 mL. of loading buffer (Laemmli, 1970) and heated to 90°C for 3 min. The proteins were separatedby electrophoresis in 10% polyacrylamide gels containing 0.1% SDS and electrically transferred onto nitrocellulose membranes(Schleicherand Schuell, pore size 0.45). The membraneswere washed in Tris-buffered salinecontaining 0.1% Tween 20 (TBS-Tween)and neutralized by a 2-h incubation at 20°C in TBSTween containing 3% skimmed milk, incubated overnight at 4°C with anti-ERK antibody (no. 691,Santa Cruz Biotechnology) diluted 1:300in TBSTween containing 1% skimmed milk, washed in TBSTween, and then incubated 1 h at 20°C with an anti-rabbit antibody conjugated to horseradish peroxidase (Amersham) diluted 1:lOOOin TBS-Tween. Finally the proteins were visualized using ECL detection system (Amersham).

RESULTS MPF is undetectable

after oocyte activation

Previous studies showed that incompetent prophase I-arrested oocytes from 12-14-day-old mice undergo premature chromosome condensation when fused to M II oocytes (Balakier, 1978). Therefore, these oocytes can be used to monitor MPF activity following oocyte activation by cell fusion with activated oocytes. We fused early parthenogenetically activated oocytes at different time points after activation with incompetent prophase I-arrested oocytes. During the first 15 min after ethanol activation oocytes were very fragile and never gave rise to any viable hybrid. Therefore, the earliest hybrids (n = 12) were obtained 20 min after activation. None of these hybrids underwent condensation of the GV chromatin indicating that 20 min after oocyte activation MPF was inactivated. Similarly, in hybrids obtained at later time points (30-100 min) after activation (n = 47), the GV remained intact. We thus concluded that MPF is inactivated within the first 20 min postactivation. This observation is in agreement with the observation that histone Hl kinase activity (biochemical measurement of MPF activity) declines in activated oocytes when anaphase II begins, ie before 15 min post-activation (Sziillijsi et al, 1993). CSFafter mouseoocyte activation

643

CSF but not MPF is present in freshly activated oocytes The CSF activity in parthenogenetically activated oocytes was examined by fusion with parthenogenetically activated eggs entering the first mitotic division as previously described (Kubiak et al, 1993). The cytoplasm of M II-arrested oocytes contains CSF which stabilizes MPF in one-cell mitotic embryos. However, MPF stabilization takes place only when MII-arrested oocytes are fused with parthenogenetic one-cell embryos, and not when fused to zygotes (Zernicka-Goetz et al, 1995). In contrast to zygotes, parthenogenetic one-cell mitotic embryos do not contain an activity leading to CSF inactivation. Therefore, in our experiments, CSF activity in activated oocytes was assayed by fusion with parthenogenetic one-cell embryos entering the first embryonic mitosis (fig 1). We expected that hybrids obtained in such experiment will either arrest in M-phase or cleave and enter interphase of the second embryonic cell-cycle. If activated oocytes had CSF activity they should stabilize the MPF preserit in mitotic embryos. If these oocytes did not contain CSF activity, the hybrids should complete mitosis and enter interphase within l-2 h, as control mitotic embryos. Most hybrids obtained 20 min post-activation (92.3%) underwent cleavage and formed two-cell embryos (fig 2) while the remaining 7.7% were arrested in M-phase, indicating that in these hybrids MPF was stabilized by CSF present in the activated oocyte (fig 2). Surprisingly, when one-cell parthenogenotes were fused with oocytes 30-60 min after activation, the proportion of M-phase arrested hybrids increased to a maximum of 59% 50-60 min post-activation (fig 2). 70-80 min after activation the number of hybrids remaining in M-phase started to decrease and only 9.5% of oocytes fused 90-100 min after activation induced a M-phase arrest (fig 2). In a control experiment, 88% (n = 17) of hybrids between onecell parthenogenetic embryos entering mitosis and M II oocytes became arrested in M-phase (fig 2, M II). All control one-cell parthenogenetic embryos (n = 21) completed mitosis and entered the second mitotic interphase. Finally, oocytes activated shortly before fusion either extruded the second polar body (62%; II = 24) or underwent immediate cleavage (37%; n = 24) and entered the first mitotic interphase.

MAP kinase remains active in M-phase-arrested hybrids It is known that active MAP kinase is required for CSF activity (Verlhac et al, 1996). Therefore, we tested whether MAP kinase remains active in MCiemerych and Kubiak

644

Biology of the Cell 91 (1999) 641-647

activated oocyte

nitotic

onacell entxyo

ni totic onwell entnyo

fusion

fusion

24 hours culture

Interphase @

MII - arrested oocyte

@

24 hours culture

M-phasearrest

@)

IM-phasearrest

Fig 1. Fusion partners and major hybrid outcome after 24 h of culture are represented. A. Experimental hybrids between parthenogenetic, mitotic one-cell embryos and activated oocytes. B. Control hybrids between parthenogenetic, mitotic onecell embryos and M H-arrested oocytes.

phase-arrested hybrids obtained by fusion between freshly activated oocytes and one-cell parthenogenetic embryos entering mitosis. We examined the electrophoretic mobility of ERKl and ERK2 in hybrids which were cultured for 24 h after fusion. The activity of these kinases correlates with a retardation in their gel-mobility caused by phosphorylation. In M-phase-arrested hybrids, we detected the slow phosphorylated forms of ERKl and ERK2 (fig 3, lane 1). In hybrids which completed cleavage division and entered interphase we detected the rapid non-phosphorylated forms of ERKl and ERK2 (fig 3, lane 2). I’hus, the MAP kinases remain active in hybrids arrested in M-phase and are inactivated upon transition to interphase.

DISCUSSION In this paper we examined the dynamics of CSF activity at the beginning of the first cell cycle of one-cell mouse embryos issued from the parthenoCSF after mouseoocyte activation

genetic activation. Since CSF stabilizes MPF in M IIarrested oocytes it was assumed that CSF inactivation is the primary cause of MPF inactivation during the completion of the second meiotic division (Masui and Markert, 1971; Newport and Kirshner, 1984; Sagata et al, 1989; Lorca et al, 1991). Unexpectedly, subsequent studies have suggested that after Xenoptls oocyte activation the MPF activity disappears prior to the CSF inactivation (Watanabe et al, 1991). This sequence of events is an apparent paradox. These observations prompted us to investigate the temporal patterns of MPF and CSF early after mouse oocyte parthenogenetic activation. Our results on parthenogenetic mouse one-cell embryos only partially agree with the results obtained with Xenopus embryos by Watanabe et al (1991). Moreover, differences we found in the profile of the CSF activity in the mouse embryo uerstls Xenopus embryo enable us to postulate a different interpretation of events early post-activation at least in the mouse. Our results show that in mouse oocytes the Ciemerych and Kubiak

Biology of the Cell 91 (1999) 641647

645

n=l7

n=29#* n=53

n=21 *

M II

20

3040

50-60

minutes

70-80

90-100

after activation

Fig 2. Proportion of M-phase-arrested hybrids among hybrids obtained after fusion with parthenogenetic, mitotic one-cell embryos and oocytes at different time points after activation. Data analyzed by ~2 test (two by two tables, Yates correction). #, results which differ statistically; P < 0.02; *, results which differ statistically, P < 0.004.

1

2

C”.., .’ , (

‘e. l”lj’<

.*,‘. _,

,S‘, .

=

z

= ERKl = ERK2

Fig 3. Western blot of MAP kinases ERK 1 and ERK 2 showing that they remain in the active, phosphorylated state (upshift) in hybrids arrested in the M-phase (lane 11, and in the inactive, dephosphorylated state (downshift) in hybrids which entered the interphase (lane 2).

MPF activity diminishes dramatically within 20 min after oocyte activation. Surprisingly, after mouse oocyte activation, CSF is inactivated in a two-step manner. The first drop of CSF activity is detectable 20 min after oocyte activation. Although we could not assay CSF in oocytes before the first 20 min post-activation, at this earliest testable time point following activation the oocytes showed the CSF activity dramatically lower when compared to the activity in M II-arrested oocytes. This result CSF after mouse oocyte activation

shows that CSF is indeed low directly after the complete inactivation of MPF taking place within 15 min post-activation (as measured by histone Hl kinase activity)( Szlilliisi et al 1993). It suggests that contrary to the interpretation of Watanabe et al (1991) for Xenupus activated oocytes, in the mouse oocytes CSF could be inactivated before MPF inactivation as postulated by Masui and Markert (1971). After the initial decrease we observed a transient increase in CSF activity at 30-40 min post-activation. Final inactivation of CSF started 70-80 min post-activation. Thus, in activated mouse oocytes the CSF activity oscillates. We do not know, however, whether the same profile of CSF activity appears also in fertilized mouse oocytes. This cannot be easily examined by the cell-fusion assay using embryos within minutes after fertilization because of the high asynchrony of the process of fertilization comparing to the parthenogenogenetic activation. Since the CSF activity is attributed to ERK-type MAP kinase activity triggered by Mos (Verlhac et nl 1996), we examined the state of the activity of these kinases in hybrids. In the M-phase-arrested hybrids, we detected the phosphorylated, active forms of ERK 1 and ERK 2 MAP kinases, while in the inactivated ones we detected inactive forms of these enzymes. These results are consistent with the role of ERK-type MAP kinases in the CSF activity. Apparently, the introduction of mitotic components, most likely MPF (since CSF is not present in the late one-cell parthenogenetic embryo), into parthenogeneticaly activated oocytes blocks the inactivation of MAP kinases and presumably the degradation of Mos. This point requires further detailed studies. The precise relationship between the MAP kinases activity and CSF inactivation is not clear. Abrieu and colleagues (1996) found by careful sampling (every 2-3 min during a period of 60 min following addition of a constitutively active mutant of Ca*+/calmodulin dependent protein kinase II) that in cell-free Xenopus eggs extracts MAP kinase remains active at the time points when MPF is inactivated. Thus, despite ERK2 MAP kinase (Xenopus oocytes do not have ERK 1 form of MAP kinase) remains continuously active and phosphorylated on the critical Thr/Tyr residues, the CSF activity seems to be inactivated. A similar pattern of uninterrupted ERK-type MAP kinases activity is observed also in mouse oocytes upon activation where these kinases remain fully active for at least 2 h (see eg Verlhac et al 1994; Kalab et al, 1996). Our data showing the low CSF activity while ERK-type MAP kinases remain fully active suggest that an unknown factor(s) regulate(s) the CSF activity during this period independently from ERK-type MAP kinases. It could be achieved if the CSF activity of Ciemerych and Kubiak

646 the Mos/.../ERK-type MAP kinases pathway is transmitted to its target (MPF?) by yet unidentified intermediate (potential MAP kinase substrate) inactivated transiently in a manner independent from these MAP kinases. Transient inactivation of CSF upon oocyte activation could therefore imply a down-regulation of Mos pathway downstream from ERK-type MAP kinases. Since the calcium transients are triggered upon mouse oocyte activation (Cuthbertson et al, 1985) it is possible that a calcium-dependent mechanism plays a role in modulation of CSF upon oocyte activation. This is in agreement with the role postulated by Abrieu et al (1996) for Caz+/calmodulin dependent protein kinase II in CSF and MPF inactivation in Xenopus egg extracts. Our data suggest that the transient CSF inactivation proceeds independently from a long-term inactivation of the Mos patway. The latter includes Mos degradation and dephopsphorylation of MAP kinases. The alternative for the complex regulation of the CSF activity is an involvement of an independent, parallel pathway. Gabrielli et al (1993) suggested that cdk2 kinase could play a role in the CSF activity in maturing Xenopus oocytes. However, the role of this kinase in the CSF activity is still not clear in Xenupus (Furuno et al, 1997), and there are no data available concerning mouse oocytes. What are the molecular bases of the increase of CSF activity after its transient inactivation following oocyte activation? The increase of CSF activity observed 30-60 min. after mouse oocyte activation takes place in the cytoplasm where Mos protein is still present and ERK-type MAP kinases are activated (Weber et al, 1991; Verlhac et al, 1994). The biological role of this particular period of the first mitotic cell cycle is to provide suitable environment for the nuclear transformation of the sperm nucleus, formation of female pronucleus and progressive reformation of the microtubule cytoskeleton of the one-cell embryo (%611&i et al, 1993; Verlhat et al, 1994). This is linked to the action of specific kinases phosphorylating some substrates like the 35-kDa protein complex (Szdlliisi et aZ, 1993). One of the consequences is probably MAP kinase activity maintained at high level at least during the first 2 h after oocyte activation (Sziilliisi et al, 1993). Therefore, the presence of the CSF activity during this period might result from the maintenance of MAP kinase activity necessary to fulfill the roles mentioned above. Reactivation of the CSF activity following MPF inactivation in mouse oocytes has also been illustrated by the abortive activation of freshly ovulated M II oocytes. These oocytes in response to parthenogenetic stimulus undergo second meiotic division but then MPF is reactivated and stabilized CSFafter mouseoocyte activation

Biology of the Cell 91 (1999) 641-647

while oocytes enter the third meiotic-like M-phase (M III) (Kubiak, 1989). Thus, the stable activity of MPF in M III-arrested oocytes clearly depends on CSF activity which must be reactivated after anaphase II. It was shown that the choice between M III and interphase depends on the age of oocytes (Kubiak, 1989) and is also linked to the quantity of free calcium liberated upon activation procedure (Vincent et al, 1992). This supports our hypothesis that the transient CSF inactivation allowing a drop in MPF activity is triggered by an increase in free cytosolic calcium. The complete molecular identity of the CSF pathway and the mechanism of its action on MPF remain unknown. We suggest that one of the reasons for the difficulty in biochemical characterization of CSF could be due to its complex character including both the Mos- and MAP kinase-mediated phosphorylation in parallel with a postulated here downstream, potentially calcium-sensitive, step and/or parallel activity independent from ERK-type MAP kinases. We believe that our description of the fluctuations in the CSF activity following mouse oocyte activation provides important information to facilitate the molecular dissection of all components of CSF, mechanisms of their action on MPF, and mechanisms of their inactivation upon oocyte activation.

ACKNOWLEDGMENTS We are grateful to Dr Bernard Maro and ProfessorAndrzej K Tarkowski for their support during experimental work, stimulating discussionsand critical reading of the manuscript. We thank Dr Yoshio Masui for valuable comments on our manuscript and Drs Zbigniew Polar&i for help in statistical analysis of our data and Jacek Gaertig for English corrections and critical reading of the manuscript. This work was supported by grants from: ARC (1361)to JZK, the State Committee for Scientific Research (KBN) to the Department of Embryology, University of Warsaw (6PO4C 048 15), short fellowships from FrenchPolishScientific and TechnologicalCooperationJoint Projects(No 6284).

REFERENCES Abrieu A, Lorca T, Labbe J-C, Morin N, Keyse S and Doree M (1996) MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts. J Cell Sci 109,239-246 Balakier H (1978) Induction of maturation in small oocytes from sexually immature mice by fusion with meiotic or mitotic cells. Exp Cell Res 112, 137-141 Ciemerych MA and Kubiak JZ (1998) Cytostatic activity devel;;;-t;;ing meiosis I in oocytes of LT/Sv mice. Dm Bid 200, Cuthbertson KSR, Whittingham DG and Cobbold PM (1981) Free Ca++ increases in exponential phases during mouse oocyte activation. Nature 294,754-757 Cuthbertson KSR (1983) Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol. I Exp Zool 226.311-314

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Cuthbertson KSR and Cobbold PM (1985) Phorbol ester and sperm activate mouse oocyte by inducing sustained oscillations in cell Cat+. Nature 316,541-542 Czolowska R, Waksmundzka M, Kubiak JZ and Tarkowski AK (1986) Chromosome condensation activity in ovulated metaphase II mouse oocyte assayed by fusion with interphase blastomeres. J Cell Sci 84,129-138 de Pennart H, Houliston E and Maro B (1988) Post-translational modifications of tubulin and the dynamics of microtubules in mouse oocytes and zygotes. Biol Cell 64,375-378 Furuno N, Ogawa Y, Iwashita J, Nakajo N and Sagata N (1997) Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase. EMBO 116,3860-3865 Gabrielli BG, Roy LM and Maller JL (1993) Requirement for Cdk2 in cytostatic factor-mediated metaphase II arrest. Science 259,1766-1769 Haccard 0, Sarcevic B, Lewellyn A, Hartley R, Roy L, Izumi T, Erikson E and Maller JL (1993) Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science 262,1262-1265 Kilmartin JV, Wright B and Milstein C (1982) Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. 1 Cell Bio293,576-582 Kalab I’, Kubiak JZ, Verlhac M-H, Colledge WH and Maro B (1996) Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation. Development 122, 1957-1964 Kubiak JZ (19891 Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest. Den Bid 136,537-545 Kubiak JZ, Weber M, de Pennart H, Winston N and Maro B (1993) The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF. EMBO J 12,3773-3778 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Lee MG and Nurse P (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Ndure 327,31-35 Lorca T, Galas S, Fesquet D, Devault A, Cavadore J-C and Do&e M (1991) Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca2+-calmodulin dependent event. EMBO ] 10,2087-2093 Lorca T, Cruzalequi FH, Fesquet D, Cavadore J-C, Mery J, Means A and Do& M (1993) Calmodulin-dependent protein kinase II mediates Ca2+-dependent inactivation of MPF and CSF upon the fertilization of Xenopus eggs. Nature 366,270-273 Maro B, Johnson MH, Pickering SJ and Flach G (1984) Changes in actin distribution during fertilisation in mouse eggs. J Embryo1 Exp Morphol81,211-237 Masui Y and Markert CL (1971) Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zoo1 117,129-146 Miyazaki S (1988) Inositol 1,4,5-trisphosphate-induced calcium release and guanine nucleotide-binding protein-mediated periodic calcium rises in golden hamster eggs. J Cell Biol 106,345-353 Murray AW, Solomon MJ and Kirshner MW (1989) The role of

CSF after mouse oocyte activation

cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature 25,28&286 Nebreda AR and Hunt T (1993) The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extract of Xenopus oocytes and eggs. EMBCJ ]12,1979-1986 Newport JW and Kirschner MW (1984) Regulation of the cell cycle during early Xenopus development. Cell 37,731-742 Posada J and Cooper JA (1992) Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science 255,212-215 Posada J, Yew N, Ahn NG, Vande Woude GF and Cooper JA (1993) Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol Cell Biol 13, 2546-2553 Sagata N, Watanabe N, Vande Woude GF and Ikawa Y (1989) The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 342, 512-518 Szollosi MS, Kubiak JZ, Debey I’, de Pennart H, Szollosi D and Maro B (1993) Inhibition of protein kinases by B-dimethylaminopurine accelarates the transition to interphase in activated mouse oocytes. J Cell Sci 104,861-872 Verlhac M H, de Pennart H, Maro B, Cobb MH and Clarke HJ (1993) MAP kinase becomes stably activated at metaphase and is associated with microtubuleorganizing centres during meiotic maturation of mouse oocytes. Dev Bid 158, 330-340 Verlhac M-H, Kubiak JZ, Clarke HJ and Maro B (1994) Microtubule and chromatin behaviour follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 120,1017-1025 Verlhac M-H, Kubiak JZ, Weber M, Geraud G, Colledge WH, Evans MJ and Maro B (1996) Mos is required for MAP kinase activation and is involved in microtubule organisation during mouse meiosis. Development 122,815-822 Vincent C, Cheek TR and Johnson MH (1992) Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium. J Cell Sci 103,389-396 Watanabe N, Vande Woude GF, Ikawa Y and Sagata N (1989) Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs. i%zture 342 (6249) 505-511 Watanabe N, Hunt T, Ikawa Y and Sagata N (1991) Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs. Nature 352,247-248 Weber M, Kubiak JZ, Arlinghaus RB, Pines J and Maro B (1991) c-))IOS proto- oncogene product is partially degraded after release from meiotic arrest and persists during interphase in mouse zygotes. Den Bioll48,393-397 Winston N (1997) Stability of cyclin B protein during meiotic maturation and the first meiotic cell cycle division in mouse oocyte. Biol Cell 89, 211-219 Zemicka-Goetz M, Ciemerych MA, Kubiak JZ, Tarkowski AK and Maro B (1995) Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs. J Cell Sci 108,469-474 Received

15 July 1999; accepted

9 September

1999

Ciemerych and Kubiak