Involvement of Carbohydrate Residues of the Zona Pellucida in In Vitro Sperm Recognition in Pigs and Cattle

The zona pellucida (ZP), which surrounds the mammalian oocyte, plays roles in species-selective sperm–oocyte interactions. In pigs and cattle, the ZP consists of ZP2, ZP3, and ZP4. Nonreducing terminal β-galactosyl (Gal) residues of neutral N-linked carbohydrate chains of the ZP are necessary for porcine sperm–ZP binding, and nonreducing terminal α-mannosyl (Man) residues of high-mannose-type chains are necessary for bovine sperm–ZP binding. Acrosome-intact porcine sperm prefer β-Gal, whereas acrosome-intact bovine sperm prefer α-Man, as shown using glycolipid analogues. The major N-linked chains of recombinant porcine and bovine ZP glycoproteins expressed using the baculovirus-Sf9 cell expression system are pauci- and high-mannose-type chains that are different in structure from the major neutral N-linked chains of the native porcine ZP but similar to those of the native bovine ZP. Porcine and bovine ZP3/ZP4 complexes coexpressed in Sf9 cells bind to bovine sperm but not to porcine sperm. Hybrid complexes consisting of native porcine ZP4 and recombinant porcine ZP3 bind to porcine sperm, whereas complexes consisting of native porcine ZP3 and recombinant porcine ZP4 do not bind to porcine sperm. These data indicate that the sugar preference of sperm is consistent with the nonreducing terminal residues of N-linked chains of sperm binding-active ZP glycoproteins and suggest that, in the in vitro assay system, the nonreducing terminal sugar residues are essential for species-selective recognition of sperm in pigs and cattle.