MAPPING THE MOUSE ZP3 COMBINING SITE FOR SPERM BY EXON SWAPPING AND SITE-DIRECTED MUTAGENESIS

During fertilization in mice, sperm bind to mouse ZP3 (mZP3), a M(r) a pproximate to 83,000 glycoprotein present in the ovulated egg extracel lular coat, or zona pellucida. Sperm recognize and bind to specific se rine/threonine-linked (O-linked) oligosaccharides present at the mZP3 combining site for sperm. Binding to mZP3 induces sperm to undergo a f orm of exocytosis, the acrosome reaction. To map the mZP3 combining si te for sperm, we examined the effect of exon swapping and site-directe d mutagenesis on the glycoprotein's two activities, sperm binding and induction of the acrosome reaction. Stably transfected embryonal carci noma cell lines were established that synthesized recombinant glycopro teins and secreted them into the culture medium. The glycoproteins wer e partially purified from culture medium and assayed for sperm-binding and acrosome reaction-inducing activities. Results of these assays su ggest that glycosylation of one or more of five serine residues, clust ered together in a polypeptide region encoded by mZP3 gene exon 7, is required for activity. Interestingly, this polypeptide region exhibits considerable sequence divergence during evolution and may be related to the proposed role for oligosaccharides in species-specific gamete a dhesion during mammalian fertilization.