Studies on the epitope of pig zona pellucida recognized by a fertilization-blocking monoclonal antibody

Heat solubilized pig zona pellucida (pZP) was separated into four major glycoprotein families (pZP1, pZP2, pZP3, pZP4). Cloning of a full-length cDNA coding for pZP1 revealed that the coding sequence for pZP4 was immediately followed by the sequence for the amino-terminal residue of pZP2 and that they shared a significant similarity with mouse and human ZP2, which are considered to serve as a secondary sperm receptor in sperm-zona interactions. One of the monoclonal antibodies, mAb-5H4, produced against pZP4 inhibited the binding of boar and human spermatozoa to each of the homologous oocytes. The epitope recognized by mAb-5H4 was determined to be present on a 10 amino acid sequence of pZP1(50-59) by using epitope mapping and analysis of chain flexibility of pZP4. A synthetic 18mer peptide corresponding to pZP1(50-67) reacted with mAb-5H4, and mouse antisera raised to the synthetic 18mer peptide recognized intact pig zona pellucida and strongly inhibited the fertilization of pig oocytes with boar spermatozoa in vitro. These results suggest that the pZP4 peptide (50-59) identified as an epitope for mAb-5H4 could be a promising candidate in the development of a contraceptive vaccine.