The long range objective of the proposed research is the isolation and characterization of glycoproteins comprising the mammalian egg envelope, the zona pellucida, with the goal of correlating structures of specific zona macromolecules with reproductive function. Sperm-egg interactions in mammalian species are known to involve recognition of carbohydrate ligands in the zona pellucida by complementary receptor molecules on the sperm surface. The scientific goal of this application is to identify and characterize the biologically active carbohydrate chains of a sperm adhesive glycoprotein, ZP3alpha, of the pig zona pellucida. Biochemical studies will profile oligosaccharide populations at individual glycosylation sites. Oligosaccharides released from purified glycopeptides by chemical or enzymatic cleavage will be separated by high pH anion exchange chromatography and structurally characterized by mass spectrometric methodologies to define site-specific carbohydrate populations. The contribution of saccharides to the sperm adhesive properties of ZP3alpha will be probed by assaying the ligand activity of ZP3alpha glycopeptides as well as ZP3alpha preparations subjected to stepwise enzymatic deglycosylation. Ligand activity will be assessed by competitive sperm-zona attachment assays as well as by a competitive ligand binding assay employing immobilized boar sperm membrane vesicles as the source of receptors and biotinylated ZP3alpha as tracer. Collectively, the data obtained will yield new and important information on the structure and function of mammalian zona glycoproteins, particularly in regard to their carbohydrate moieties. Considering common mechanisms of gamete interactions across species and the immunological and structural similarities of human and pig zona proteins, such knowledge will contribute to a better understanding of human reproductive biology as well as the eventual development of novel approaches to prefertilization contraception and the diagnosis and treatment of infertility.