The overall goal of this proposal is to determine the specific carbohydrate determinants within the zona pellucida that are recognized by mammalian sperm during fertilization. A number of biochemical and cell biological approaches will be used to precisely determine the exact nature of the oligosaccharides on the glycoprotein ZP3, which are necessary for sperm-zone adhesion and the induction of acrosomal exocytosis. Quantitative assays using solubilized and 125I-labeled zone pellucida glycoproteins to characterize the interaction between ZP3 and its complementary receptor(s) on the mouse sperm surface have been developed. These assays will be used to probe the specific molecular interactions involved in the recognition between sperm and egg in a manner which is not possible using previous cell binding assays. Using this new assay, defined monosaccharides, simple oligosaccharides, and complex oligosaccharides will be compared to identify potential carbohydrate moieties which are involved in sperm adhesion to the zona pellucida. Specifically, oligo-saccharides which contain terminal sugars that have been implicated in sperm-zone adhesion will be tested. By varying both the composition and the complexity of these oligosaccharides, the carbohydrate recognition domains on the sperm's ZP3 receptor will be examined. In addition, the ZP3 glycoprotein will be chemically dissected to quantify the contributions of both O-linked and N-linked oligosaccharides, as well as specific terminal monosaccharides on ZP3, in sperm-zone binding. These oligosaccharides will be initially analyzed using lectin affinity chromatography and mass spectrometry to determine the specific sugars and oligosaccharide mass in the ZP3 gly-coprotein. This research will yield important information about the composition of macromolecules involved in mammalian sperm-egg recognition and adhesion and may have implications both for the development of contraceptives and for understanding specific molecular lesions which may lead to infertility.
