Fertilization of mouse eggs initiates a phenomenon termed """"""""egg activation"""""""". Egg activation results in (1) the stimulation of cortical granule (CG) exocytosis that results in modifications of the zona pellucida (ZP) that constitute the block to polyspermy, and (2) results in the conversion of a meiotic cycle to a mitotic one (mitotic activation). By analogy to differentiated somatic cells, the sperm may initiate these events by a G protein-mediated signal transduction pathway that results in the generation of specific second messengers/ionic changes. Specific second messengers[inositol 1, 4, 5-triphosphate (IP3) or activators of protein kinase C (PKC)] elicit the ZP modifications but do not result in mitotic activation. These observations suggest that although both CG exocytosis and mitotic activation are initiated by fertilizing sperm, different second messenger systems may be generated that differentially regulate these two responses. In addition, although G proteins mediate exocytosis and mitogenic activation in somatic cells, there are in fact no conclusive data that mammalian egg activation is mediated by a G-protein-coupled signal transduction cascade. This proposal will address (1) the role of these second messengers in differentially regulating these two components of mouse egg activation, and (2) the role of G proteins in egg activation. These studies will use cell biological and biochemical approaches, including microinjection and in vitro fertilization of eggs. The modification of the ZP after fertilization, which constitutes the block to polyspermy, is thought to be mediated by the contents of the cortical granules, which are released following fertilization. There is little information, however, regarding the contents of CGs, let alone their mode of action on the ZP modification. This proposal will address the characterization and function of the first described mammalian egg cortical granule protein. These studies will use cell biological, biochemical, and molecular approaches, including metabolic labeling of eggs, immunoprecipitation and immunoblotting, preparation and screening of cDNA libraries, and isolation and analysis of cDNA clones. Results of these studies will provide new information regarding (1) the biochemical basis of egg activation, (2) the role of G proteins in mediating sperm-induced egg activation, and (3) the characterization and cloning of the first mammalian cortical granule antigen.
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