The long range objective of the proposed research is the isolation and characterization of glycoproteins of the mammalian egg extracellular coat, the zona pellucida, with the aim of correlating structures of specific zona macromolecules with reproductive function.
The specific aims of this application are concerted biochemical and biological investigations of the carbohydrate moiety of a purified Mr = 55,000 antigen (ZP3) of pig oocyte zona pellucida. Biochemical studies are directed toward the isolation of ZP3 oligosaccharides and detailed structural characterization of ZP3 lactosaminoglycans, i.e. high molecular weight oligosaccharides with repeating N-acetyllactosamine units. Biological studies seek to critically evaluate the contribution of carbohydrate to the sperm receptor activity of ZP3. Alkaline borohydride treatment of ZP3 will result in release of O-linked oligosaccharides. Subsequent hydrazinolysis of the residual glycopeptide will result in release of N-linked oligosaccharides. A purification protocol employing gel filtration chromatography will yield preparations enriched in N-and O-linked lactosaminoglycans as well as N- and O-linked oligosaccharides of more conventional structure. Structural characteristization of lactosaminoglycans will employ the application of methylation analyses, digestions with glycosidases and fast atom bombardment-mass spectrometry to the intact molecule and to fragments (core and peripheral oligosaccharides) generated by digestion with endo-beta- galactosidase. The sperm receptor activity of various ZP3 preparations will be evaluated by several methods: direct inhibition of sperm-zona attachment; binding of radiolabeled samples to boar sperm; and ability of Fab antibodies to inhibit sperm-zona attachment. ZP3 preparations to be tested include: intact ZP3; chemically and enzymatically deglycosylated ZP3; individual alpha- and beta-glycoprotein components; purified oligosaccharide fractions; and chemically denatured ZP3. Collectively, the data obtained will yield new and important information concerning the biochemical properties of zona pellucida glycoproteins and molecular mechanisms of sperm-egg interaction. In light of the immunological relatedness of pig and human zonae pellucidae, the proposed research may eventually lead to a better understanding of human reproductive biology as well as development of novel approaches to human contraception.
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