MECHANISMS OF MAMMALIAN FERTILIZATION/POLYSPERMY BLOCK

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In the mouse, a single glycoprotein, ZP3, accounts for sperm receptor and acrosome reaction inducing activities of the zona pellucia, which is an extracellular coat surrounding the egg. It is thought that fertilization stimulates exocytosis of the cortical granule contents, which modify the zona so that both its sperm receptor and acrosome reaction inducing activities are lost. We have observed that zonae isolated for phorbol ester-treated eggs retain their sperm receptor activity, but have lost the ability to induce a complete acrosome reaction; phorbol esters activate protein kinase C. To date, it has not been possible to examine what moieties of ZP3 are involved in the acrosome reaction. Using assays to monitor sperm-zona interaction and the acrosome reaciton, and employing glycopeptide and peptide mapping procedures, we will compare the properties of ZP3 isolated from untreated and phorbol ester-treated eggs, since these differences are likely to reflect those portions of ZP3 involved in the acrosome reaction. Since we have shown that the biochemical modification of the zona in phorbol ester-treated eggs is similar to that in fertilized eggs, we propose that these compounds stimulate the cortical granule reaction. We will test this hypothesis by characterizing the composition and enzymatic properties of secreted egg products in response to phorbol esters, and correlating this with a decrease in the number of cortical granules by electron microscopy. Lastly, we will examine the effects of microinjecting IP3 and guanine nucleotide analogs in stimulating early events of mammalian egg activation, since these compounds form a signal transduction pathway in conjunction with the protein kinase C. The role of these compounds in mammalian egg activation events is not known. Results of these studies will provide a wealth of new information regarding ( 1) the biochemical basis for the ZP3-mediated induction of the acrosome reaction, (2) how phorbol esters stimulate cortical granule exocytosis; this will provide a convenient system for future studies of the cortical granule reaction, and (3) the role of the phosphoinositide turnover pathway, of which guanine nucleotides, IP3, and protein kinase C activators play a central role, in early events of mammalian egg activation.