BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

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The objective of this research is to acquire a deep cellular and biochemical knowledge of the mechanisms of sperm-egg interaction during fertilization. Marine invertebrate animals, the sea urchin and the abalone, are used in this research because they are nonmammalian, inexpensive, they yield vast quantities of gametes, gamete interaction is external to the adults, it occurs synchronously in a time span of seconds, and because of the striking similarities between marine invertebrate and mammalian fertilization. The basic knowledge gained may be applied in the future to the development of novel methods of nonhormonal contraception in mammals. This proposal focuses on two phenomena of sperm-egg interaction: The mechanism by which sea urchin egg jelly binds to sperm plasma membrane receptor proteins to induce the sperm acrosome reaction; and the mechanism by which abalone sperm lysin dissolves the egg vitelline envelope by a nonenzymatic process. The basic mechanisms underlying these two phenomena of sperm-egg interaction are potential targets for nonhormonal contraception. The specific aims of this project are the following: Sea urchin sperm: To isolate the intact glycoprotein from egg jelly that induces the sperm acrosome reaction. To clone and characterize this protein and to determine if it has homologs in higher species. To characterize the inductive oligosaccharides of this egg jelly glycoprotein. To determine which sperm membrane proteins are receptors for them. To determine the amino acid sequence, function and distribution of the 6OkD protein of sea urchin sperm. The 6OkD and 2lOkD sperm membrane proteins are the receptors for the egg jelly glycoprotein which induces the sperm acrosome reaction. The 6OkD protein is homologous to human uromodulin, a protein of unknown function that is high in pregnancy urine. To clone, sequence and determine the homology and physiological role of the 2lOkD protein from sea urchin sperm membranes. Is it a calcium channel or a regulator of calcium channels? Abalone egg vitelline envelopes (VEs): To determine the ultrastructure of the VE glycoprotein fibers before and after dissolution of VE with crude sperm lysins, pure lysins, alcohol or heat. To isolate the individual 3-4 VE glycoproteins and determine which bind to sperm lysin. Is it a peptide or an oligosaccharide recognition domain that binds to lysin? Why is the reaction species specific? To clone and sequence the VE glycoproteins which bind to lysin. To completely characterize the VE ligand domains (oligosaccharide or peptide) which show species specific affinity for sperm lysin. If peptide, to perform site directed mutagenesis to determine the crucial residues. To clone, sequence and characterize the protein comprising the paracrystalline lining of the abalone sperm acrosomal granule. The results of this project are central to deepening our knowledge of the biochemistry of sperm-egg interaction in animal fertilization.