Marangos, Petros; (2004) The regulation of meiosis in mouse oocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The overall aim of the experiments presented in this thesis is to investigate the regulation of meiosis in mammalian oocytes. To investigate the role of cyclin B during progression through meiosis I to II we have made use of a cyclin B1-GFP fusion protein. Injection of cyclin B1-GFP accelerates GVBD and overrides cAMP-mediated arrest at the GV stage. Excess cyclin B can accelerate or inhibit the extrusion of PB1 in a dose-dependent manner. The distribution of cyclin B1-GFP was found to be controlled through the regulation of nuclear import and export. Within 15 minutes of GVBD, cyclin B1- GFP accumulates in the GV, presumably due to a rise in import and a decrease in export. Cyclin BL-GFP is also a tool for examining cyclin degradation that is necessary for exit from M-phase. In MI we find cyclin B destruction is necessary for progression through MI. Cyclin B destruction at Mil is stimulated by an increase in Ca2+ at fertilisation. This destruction results in an increase in the rate of cyclin B degradation. Producing Ca2+ transients during MI does not induce cyclin B degradation showing cyclin B destruction becomes sensitive to Ca2+ late in meiosis. Furthermore, we examined the role of Emil in meiosis. Emil is present in both MI and MIL By microinjecting Emil protein we found that Emil blocks polar body extrusion. By injecting morpholinos aimed against the endogenous Emil mRNA, we managed to block the maturation of oocytes at prometaphase which implies a role for Emil in MI. Emil depletion also caused the release of MII eggs from metaphase arrest. This showed that this protein may be, as MAPK, a component of the cytostatic factor, which is responsible for the arrest at MII. Finally, we examined the relationship of Ca2+ oscillations and cell cycle resumption at fertilisation. Ca2+ oscillations do not depend on normal levels of CDKl-cyclin B since they continue after CDKl activity has declined. Moreover, they are not sensitive to the MAPK inhibitor, U0126. The data demonstrate a strong correlation between Ca2+ oscillations and Pn formation. In this thesis we present a model whereby Ca2+ oscillations at fertilisation and mitosis are controlled by the nuclear sequestration of a sperm-derived Ca2+-releasing factor, such as PLCζ.

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