Control of the cell cycle during meiotic maturation of mouse oocytes

The overall aim of the work presented in this thesis is to better understand the molecular \nmechanisms underlying the progression of the meiotic division in mammalian oocytes. \nThe first goal of this project was to investigate the relative contribution of securin and \ncyclin B1 to the control of the protease separase during the indeterminate period of \nmetaphase II arrest. I found here that although there are conditions in which either \nsecurin or MPF can prevent chromosome disjunction, such as during meiosis I, securin is the predominant inhibitor of separase during metaphase II arrest. Thus, CDK1 \npharmacological inhibition as well as antibody inhibition of CDK1/cyclin B1 binding to separase, both failed to induce sister chromatid disjunction. Instead, securin morpholino knockdown induced sister chromatid separation, which could be rescued by injection of securin cRNA. \nI also examined the effect of CDK1 and MAPK activities on securin stability. Inhibition of both kinases, but not either one alone, using roscovitine and UO126, induced the \npremature destruction of securin during prometaphase of meiosis I. However, this effect \nis not dependent on securin phosphorylation, given that mutagenesis of CDK1 and \nMAPK phosphorylation sites on securin did not affect its stability. \nFinally, I found that CDK1 and MAPK inhibition also causes the premature degradation of other APC/C substrates, such as cyclin B1 and geminin. Interestingly, only the D-box containing substrates and not Ken-box substrates were subject to such effect. However, this prometaphase I instability of the D-box substrates was apparently not mediated by APC/CCdc20 or APC/CCdh1, given that Cdc20 was also targeted for destruction and morpholino-knockdown of both Cdc20 and Cdh1 failed to rescue securin stability. Also, the inhibition of SCFβ-TrCP using the dominant negative mutant of its F-box protein (β-TrCPΔ) could not restore securin levels, suggesting it is not SCFβ-TrCP-dependent. Future \nwork will explore whether this effect could be attributed to activation of some meiosis-specific \nAPC/C co-activators or the core APC/C, which was recently reported of being \nable to directly interact with the D-box containing substrates independently of Cdc20 and \nCdh1 (Passmore et al., 2003; Yamano et al., 2004).