Biochemical and functional characterization of zonadhesin: A sperm protein potentially mediating species-specific sperm-egg adhesion during fertilization

Fertilization is an important and unique process in organisms that generate offspring by sexual reproduction. Several fines of evidence support the idea that glycoproteins in the egg zona pellucida (ZP) mediate species-specific sperm-egg adhesion. In contrast, studies on the complementary molecules in spermatozoa remain controversial. Some potential candidate adhesion molecules have been identified, but none of them is supported by unequivocal evidence. A mosaic protein called zonadhesin is one of them. Zonadhesin was first found in pig sperm membrane extracts based on its ability to bind to the native ZP in a species-specific manner. The zonadhesin cDNA sequence reveals that the protein comprises several extracellular domain types found in other molecules known to mediate cell-cell interactions. Orthologs of pig zonadhesin in three other species possess domain structures similar to pig zonadhesin's, but with variations. Our overall hypothesis is that zonadhesin mediates species-specific adhesion between spermatozoa and the egg ZP. In this dissertation, I report biochemical and functional characterization of pig zonadhesin. I observed that zonadhesin forms disulfidebonded oligomers and multimers, but the pl05/p45 monomer displays a preference for binding to the ZP. Zonadhesin undergoes heterogeneous processing and is targeted to more than one physicochemical compartment in developing sperm cells. Posttranslational processing generates at least three zonadhesin components that are glycosylated with different types of oligosaccharides. Zonadhesin's oligomeric status changes during maturation of spermatozoa in the epididymis. Immunoelectron microscopy demonstrated that zonadhesin localizes to the outer acrosomal membrane and the acrosomal matrix. Moreover, I identified zonadhesin protein in eight additional species by Western blotting, immunofluorescence, or both. Zonadhesin from each of these species localizes to the apical sperm head overlying the acrosome, but the MrS of these proteins' polypeptide chains vary significantly. In addition, a bead adhesion assay was developed for future tests of zonadhesin's ability to bind to homologous or heterologous ZP. Finally, preliminary work on expression of zonadhesin domain(s) initiated future loss- and gain-of-function transgenic studies. Potential practical benefits derived from this work may include development of new contraceptives that function by blocking gamete interactions and diagnostic methods for human fertility and farm animal fecundity.