The long-term goal of this application is to elucidate the mechanisms that control egg activation in mammalian species. Mammalian eggs are ovulated arrested at the metaphase stage of the second meiosis (MIl). Egg activation entails exit from MIl and the completion of a series of morphological and biochemical changes that initiate the mitotic and, consequently, the developmental program. Fertilization induces egg activation by evoking periodical changes in the intracellular concentration of free calcium ([Ca2+]i) known as [Ca2+]i oscillations. The inositol 1, 4, 5-trisphosphate receptor (IP3R-1), a ligand-gated channel, mediates the [Ca2+]i rises at fertilization. Evidence shows that the mass, distribution and conductivity of IP3R-1 are regulated during maturation and fertilization and that this is required to establish the temporal and spatial organization of the [Ca2+]i responses at fertilization. Despite this pivotal role of IP3R-1, little is known about the molecular mechanisms that control its function in eggs. Here, we hypothesize that IP3R-1 constitutes a key regulatory locus for the organization of Ca2+ release during egg activation. To further elucidate the mechanisms that control IP3R-1 function we propose to: 1) investigate how IP3R-1 degradation is regulated during activation and how it impacts sperm-initiated [Ca2+]i oscillations; 2) determine whether IP3R-1 phosphorylation by cell cycle-associated kinases mediates the observed association between the cell cycle, IP3R-1 conductivity and [Ca2+]i oscillations; 3) examine the localization and mechanism(s) responsible for IP3R-1 redistribution and how IP3R-1 distribution/cortical cluster formation affects Ca2+ release. To address these questions we will perform [Ca2+]i monitoring, immunoblotting, immunofluorescence, microinjection of mRNAs, release of caged compounds, and mutations and tagging of IP3R-1 followed by expression in eggs/somatic cells and confocal microscopy visualization. Relevance: Given the diverse and precise regulation of IP3R-1 function during maturation, evaluation of these parameters after ovulation/maturation may provide markers with which to assess the impact of hormonal stimulation/in vitro maturation conditions on oocyte quality. Also, since abnormal Ca2+ release occurs in eggs aged after ovulation and this may lead to embryo fragmentation, elucidation of the mechanisms that control IP3R-1 function promises to improve embryo development and the success of Assisted Reproductive Technologies. ? ? ?
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