Brind, Sophie Elizabeth; (2001) The role of inositol 1,4,5-triphosphate receptors in mammalian oocytes and preimplantation embryos. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A fertilisation-induced increase in intracellular Ca2+ is responsible for initiating all of the events of egg activation. In mammals, the Ca2+ increase takes the form of a series of Ca2+ oscillations that continue for 3-4 hours. In most cells, intracellular Ca2+ is regulated by Ca2+ channels in the endoplasmic reticulum known as inositol 1,4,5-trisphosphate receptors (InsP3Rs). The aim of the experiments presented in this thesis is to investigate the role of three known isoforms of the Insp3Rs in oocyte maturation, fertilisation and early mammalian development. Western analysis revealed that the type I isoform accounts for all of the detectable InsP3R protein in unfertilised mouse oocytes. Furthermore, type I protein levels were dramatically decreased within 4 hours of fertilisation. During development to the blastocyst the level of type I did not return to prefertilisation levels and types II and III remained below our detection limit. One mechanism that leads to the inhibition of Ca2+ transients after fertilisation may be the downregulation of InsP3Rs. I examined the mechanism of this InsP3R downregulation and found that neither egg activation nor Ca2+ transients are necessary or sufficient for its stimulation. The only stimulus, besides fertilisation, that downregulated InsP3Rs was microinjection of the potent InsP3R agonist adenophostin A, suggesting that InsP3 binding is sufficient for downregulation. InsP3R downregulation was inhibited by the cysteine protease and proteasome inhibitor ALLN but not by specific proteasome inhibitors MG 132 or lactacystin. To examine the role of InsP3-induced Ca2+ release during oocyte maturation, at fertihsation and in the preimplantation embryo, I used adenophostin A to produce immature oocytes. Mil eggs and fertilised 1-cell embryos which were depleted of InsP3Rs. I found that sperm-induced Ca2+ signaling was inhibited in InsP3R-depleted Mil eggs, indicating that Ca2+ signaling at fertilisation is mediated via the InsP3R. In contrast, I found that endogenous levels of InsP3Rs are not necessary for GVBD and meiosis I of oocyte maturation or for NEED of the first cell cycle or for subsequent cell division cycles up to the morula stage. I conclude that the type I InsP3R is essential for fertilisation but that less than 5% of the endogenous receptors are necessary for the first meiotic division and the early embryonic cell division cycles. These studies suggest that the previously reported role for Ca2+ in mitosis in mammalian cells requires re-evaluation.

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