PLASMA MEMBRANE CHANGES IN FERTILIZATION

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Despite great advances in our understanding of the structure and function of receptors in somatic cells, relatively little is known about receptors involved in the cell-cell interactions that occur at fertilization. Work in a prior grant period on the sea urchin egg receptor that binds sperm established that the receptor was protease-sensitive and that its release resulted in loss of ability of sperm to bind to and fertilize these eggs. Furthermore, it was established that proteolytically-released fragments inhibited fertilization in a species-specific manner. Subsequently, it was shown that the intact receptor was a complex proteoglycan-like molecule. Evidence was also obtained that the carbohydrate chains of the receptor serve as the adhesive element of the molecule, whereas the polypeptide chain defines the species specificity of the binding process. Given the extreme insolubility and apparent high molecular weight of the intact receptor, procedures were developed to isolate fragments of it that retained species specificity. One of these fragments, presumed to be a portion of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kDa glycosylated peptide. This receptor fragment binds to sperm species specifically. The current proposal has five objectives. First, the primary structure of the intact receptor, and the sites of attachment and the structure(s) of its oligosaccharide chains will be defined. Second, having established the basic structural features of the receptor molecule, a variety of methods will be used to partially degrade it so as to define the structural basis of its species specificity, especially with respect to the role of the polypeptide backbone and the oligosaccharide chains. Third, potential additional ligands for the receptor will be identified. Although it is clear that bindin from the sperm acrosome is one ligand involved in interaction with the egg receptor, it is not known if other sperm components are involved. Fourth, the apparent activation of eggs by antibody to the 70-kDa receptor fragment will be compared with a variety of "early activation events" induced by sperm. By microinjection of inhibitors or activators of known signal transduction pathway components it should be possible to determine if these components are involved in the sperm receptor system. If primary structure analysis indicates that the receptor is a transmembrane protein, it will be reconstituted into the plasma membrane of frog oocytes or starfish oocytes by microinjection of its mRNA. Alternatively, the intact receptor will be purified, introduced into phospholipid liposomes and then fused with the plasmic membrane of starfish oocytes. Fifth, the structure of the S. purpuralus receptor will be compared with that of other sea urchin species. Initially, this will be accomplished at the level of the gene, which will provide information about primary structure. In addition, comparative studies will be carried out with a collection of antibodies, and with an egg receptor hydrolase that appears to be species specific. A comparison of the receptor structure in various sea urchin species should provide detailed insight into the molecular basis of the species-specific interaction of sperm and egg.