Switch from the meiotic to the mitotic cell cycle

The metaphase arrest in vertebrate oocytes is maintained by cytostatic factor activity (CSF; Masui & Markert 1971). CSF was first demonstrated in amphibian oocytes by transferring cytoplasm from metaphase II (M II)-arrested oocytes into one blastomere of a two-cell embryo. The injected blastomere was arrested in M phase while the non-injected blastomere continues to cleave (Masui & Markert

1971). We have demonstrated the presence of an equivalent activity in mouse M Unarrested oocytes using a cell fusion technique (Kubiak et al 1993). CSF was defined as an activity that stabilizes the M phase-promoting factor (MPF; Masui & Markert 1971). The proto-oncogene c-mos gene product (Sagata et al 1989) was the first molecule implicated in the CSF activity. Further studies have shown that Mos is the MAP kinase kinase kinase that activates the MAP kinase pathway during meiotic maturation (Posada et al 1993, Nebreda & Hunt 1993). Activation of MAP kinase involves its phosphorylation on threonine and tyrosine residues (Posada & Cooper 1992) which correlates in mouse oocytes with a shift in the electrophoretic mobility of the two isoforms of MAP kinase, ERK1 and ERK2 (for extracellular regulated kinase; Verlhac et al 1993, 1994). After activation of M II oocytes, MAP kinase activity drops progressively during 3—4h post-activation (Verlhac et al 1994). Mos is destroyed after MPF inactivation when MAP kinase activity drops (Weber et al 1991).

MPF is heterodimer composed of a catalytic subunit, the p34cdc2 kinase, homologue of the fission yeast cdc2 gene product, and a regulatory subunit, cyclin B. 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, Winston 1997) suggesting that CSF only partially inhibits the cyclin B degradation machinery in this species. The inactivation of CSF upon oocyte activation would facilitate proteolysis of cyclin B (Kubiak et al 1993, Winston 1997). However, CSF inactivation upon oocyte activation has never been observed directly and was only anticipated from the behaviour 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 Ca2+ (Miyazaki 1988) and leads to the release of the meiotic arrest by inactivation of MPF (Lorca et al 1993) and CSF (Watanabe et al 1989). However, the detailed analysis of the dynamics of MPF and CSF inactivation in Xenopus oocyte has led to an apparent paradox: CSF activity was still detectable after MPF inactivation (Watanabe et al 1991). Does CSF inactivation follow the inactivation of MPF? Such a sequence of events contradicts, however the role of CSF in stabilization of MPF. Degradation of Mos and inactivation of MAP kinases and their substrates (e.g. p90rsk) also follow inactivation of MPF in mouse oocytes (Weber et al 1991, Verlhac et al 1994, Kalab et al 1996). Is therefore CSF inactivation parallel to inactivation of the MAP kinase pathway? To find the answer to this question we investigated the dynamics of CSF activity after mouse oocyte activation using a biological assay based on a cell fusion method.

Parthenogenetic one-cell embryos were fused very soon after an activating treatment with parthenogenetic one-cell embryos entering first mitosis. We expected that hybrids obtained in such experiments would either arrest in M-

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