Identification of Boar Sperm Ligands Bound to the Protein (pZP1) of Pig Zona Pellucida.

In previous studies we found that pig zona pellucida glycoprotein pZP1 may act as the secondary sperm receptor during penetration into the zona pellucida. In this study, a recombinant pZP1(1-198) protein (r-pZP1) was produced to examine the direct binding of pZP1 to spermatozoa. When boar spermatozoa were co-cultured with pig cumulus oophorus in TC199 medium containing r-pZP1 proteins, r-pZP1 bound to the spermatozoa invaded the cumulus oohorus. The binding site was in the acrosomal region, but no sperm binding of r-pZP1 was observed when they were incubated in the medium without cumulus oophorus. This suggests that the cumulus cells played a role in exposing the ligand molecules for r-pZP1 in the acrosomal region. When boar spermatozoa were incubated with pig cumulus-oocyte complex in the medium containing r-pZP1, r-pZP1 bound to the spermatozoa adhered to the zona pellucida in the postnuclear region and the tail. r-pZP1 also bound to human spermatozoa pretreated with Ca++ ionophore A23187 and the binding site migrated from the equatorial region to the tail during incubation. Western blot analysis with crude extracts of boar spermatozoa revealed that three molecules, Mr 55K, Mr 40K and Mr 25K, reacted to r-pZP1.