Structural Significance of N-glycans of the Zona Pellucida on Species-selective Recognition of Spermatozoa Between Pig and Cattle

The zona pellucida that surrounds the mammalian oocyte plays a role in species-selective sperm-egg interactions. In the pig and cattle, the zona pellucida consists of ZPA, ZPB and ZPC. Sperm binding activity of porcine zona glycoproteins is conferred by tri- and tetra-antennary complex-type in cattle it is conferred by a high-mannose-type chain o f f ive mannose residues. Non-reducing terminal residues of these N-linked chains, beta-galactosyl residues in pig and alpha-mannosyl residues in cattle, are involved in the binding of zona glycoproteins to respective spermatozoa. The major N-linked chains of recombinant porcine ZPB expressed using the baculovirus-Sf9 cell expression system are pauci- and high-mannose-type chains that are different in structure to the major neutral N-linked chains of the porcine zona but similar to those of the bovine zona. The mixture of porcine ZPB/ZPC co-expressed in Sf9 cells binds to bovine sperm but not to porcine sperm, indicating an essential role of the N-linked chains in species-selective recognition of sperm in pig and cattle. Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduce the sperm-binding activity of the ZPB/ZPC mixture, while a similar mutation at Asn333 has no effect on binding. These results coincide with our previous report that tri- and tetra-antennary complex-type chains are localized at Asn220 in native porcine ZPB and suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.