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 Przedmiescie 26/‘28, 00-927 Warsaw, Poland; b Biologie 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 post- activation 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 Mar- kert, 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 threo- nine 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 Biology of the Cell 91 (19991 641-647

kinase, ERK 1 and ERK 2 (for extracellular regu- In this study we investigate the dynamics of lated kinase 1 and 2; Verlhac et al, 1993, 1994). Acti- CSF activity after mouse oocyte activation using a vation of MAP kinases in mouse oocytes takes biological assay based on cell fusion method. We place after activation of MPF and germinal vesicle show that CSF activity fluctuates after oocyte acti- breakdown (GVBD) (Verlhac et al, 1993). It seems vation. Inactivation of CSF proceeds in two steps: that CSF develops already during the first meiotic first, CSF is transiently downregulated by a mech- M phase, and is transiently inactivated at anaphase I anism independent from Mos degradation and (Ciemerych and Kubiak, 1998). After activation of MAP kinase inactivation to allow exit from the M M II oocytes, MAP kinase activity drops progres- II arrest. Second, the disappearance of CSF activ- sively during 34 h post-activation (Verlhac et aI, ity after the transition to the first embryonic cell 1994). Mos is also destroyed after MPF inactivation cycle requires inactivation of the MAP kinase when MAP kinase activity drops (Weber et al, 1991). pathway.

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.

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

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 pro- vokes a drop in CSF activity. However, there are no studies showing the profile of CSF inactivation in mammalian oocyte.

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 hyaluroni- dase (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 phyto- haemagglutinin (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 Czo- lowska et al (1986). PEG-treated pairs were cultured in M2 medium.

CSF after mouse oocyte activation Ciemerych and Kubiak

Biology of the Cell 91 (1999) 641-647 643

lmmunofluorescence CSF but not MPF is present in freshly activated oocytes Hybrids were fixed and processed for immunofluores-

cence as previously described (Maro et al, 1984 modified 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 scanning confo- cal microscope.

lmmunoblotting

Five hybrid cells were 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 separated by electrophoresis in 10% polyacrylamide gels containing 0.1% SDS and electrically transferred onto nitrocellulose membranes (Schleicher and Schuell, pore size 0.45). The membranes were washed in Tris-buffered saline containing 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 1:300 in TBS- Tween containing 1% skimmed milk, washed in TBS- Tween, and then incubated 1 h at 20°C with an anti-rab- bit antibody conjugated to horseradish peroxidase (Amersham) diluted 1:lOOO in 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. Dur- ing 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 post- activation. 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).

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 con- tains 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). Surpris- ingly, 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 one- cell 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 M-


644

nitotic activated oocyte onacell entxyo

Biology of the Cell 91 (1999) 641-647

ni totic MII - arrested oocyte onwell entnyo

fusion

24 hours culture

fusion

24 hours culture

Interphase @ @ 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 one- cell 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 retar- dation 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 partheno-

genetic activation. Since CSF stabilizes MPF in M II- arrested 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). Unexpect- edly, 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). More- over, 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


Biology of the Cell 91 (1999) 641647 645

n=l7

n=29#*

n=53

n=21 *

M II 20 3040 50-60 70-80 90-100

minutes 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. .*,‘. ,S‘, ( l”lj’< _, .

= = ERKl z = 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

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 can- not 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 par- thenogeneticaly 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 sam- pling (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 unin- terrupted 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


646 Biology of the Cell 91 (1999) 641-647

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 mod- ulation 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 lat- ter includes Mos degradation and dephopsphoryla- tion 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 pro- gressive reformation of the microtubule cytoskele- ton of the one-cell embryo (%611&i et al, 1993; Verl- hat 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 mainte- nance 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 illus- trated 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

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 Professor Andr- zej K Tarkowski for their support during experimental work, stimulating discussions and critical reading of the manuscript. We thank Dr Yoshio Masui for valuable com- ments 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 French- Polish Scientific and Technological Cooperation Joint Pro- jects (No 6284).

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Transient reactivation of CSF in parthenogenetic one-cell mouse embryos

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