Polypeptide backbone derived from carboxyl terminal of mouse ZP3 inhibits sperm-zona binding

For mammalian organism, fertilization begins with species-specific recognition between sperm and egg, a process depending upon egg zona pellucida glycoproteins and putative sperm interacting protein(s). In mouse, zona pellucida glycoprotein ZP3 is believed to be the primary receptor for sperm and inducer of sperm acrosomal reaction, and its function has been attributed to the specific O-linked oligosaccharides attached to polypeptide backbone. While lots of reports have focused on the role of ZP3's oligosaccharides in fertilization, there are few concerning its polypeptide backbone. To investigate whether mZP3 polypeptide backbone is involved in sperm-egg recognition, three partially overlapping cDNA fragments, together covering entire mouse ZP3, were cloned, expressed and purified under denaturing condition. Although all three refolded proteins possess native conformation, only one derived from the carboxyl terminal showed inhibitory effect to the sperm-zona binding during in vitro fertilization. This phenomenon could not be explained by enhanced acrosomal exocytosis rate, in that the acrosomal reaction assay demonstrated its inability to induce the acrosomal reaction. Our results suggest that the carboxyl terminal of mZP3 polypeptide backbone interacts with sperm and such interaction plays a significant role in sperm-zona binding, ultimately successful fertilization.