FitzHarris, David Greg; (2003) Cell cycle control of endoplasmic reticulum structure and Ca2+ -release in the mouse oocyte and early embryo. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In mammals, fertilisation triggers a series of intracellular Ca2+ transients which are responsible for egg activation and completion of meiosis. These oscillations are generated by InsP3-induced release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ oscillations last for 3-4 hours in mouse, ceasing at the time of pronucleus formation. The subsequent breakdown of the pronuclei (NEBD) at mitosis entry is accompanied by the resumption of Ca2+ oscillations. The experiments presented in this thesis examine the relationship between ER structure and Ca2+ release in the mouse oocyte and early embryo, and investigate the role of Ca2+ release m mitosis. Using the ER-specific marker Dil, we report that germinal vesicle breakdown is associated with a dramatic microtubule-dependent redistribution of ER to the region surrounding the metaphase-I spindle. ER remains tightly packed around the spindle, during centro-cortical migration. The formation of clusters of ER in the oocyte cortex occurs around the time of polar body formation, and coincides with increased responsiveness of InsP3-mediated Ca2+ release. The decrease in cdkl-cyclin B activity which occurs following activation is both necessary and sufficient for the subsequent disappearance of ER clusters, and corresponds with diminished Ca2+ release in response to InsP3. Cortical ER clusters do not re-appear following NEBD, rather ER accumulates around the mitotic spindle. NEBD is associated with increased responsiveness of Ca2+ release both in fertilised and parthenogenetic embryos. The role of Ca2+ in mitosis was examined. Mitotic Ca2+ transients are dispensable since InsP3-receptor-downregulation and Ca2+ -chelators prohibit mitotic Ca2+ transients without affecting the first embryonic division. Microinjection of a fluorescent marker into one pronucleus reveals that nuclear membrane permeablisation begins prior to initiation of mitotic Ca2+ signals. The subsequent cessation of mitotic oscillations precedes the formation of nuclei in the two-cell embryo. No Ca transients are detected during the second mitotic division. These data demonstrate dynamic microtubule and cell cycle dependent ER reorganisations in meiosis and mitosis, in which clustering of ER in the cortex or around the spindle is associated with increased responsiveness of InsP3-releasable Ca2+ stores. Additionally, the results presented suggest that global mitotic Ca2+ transients are triggered by NEBD, rather than being the cause.

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