Species specificity of human and murine anti-ZP3 synthetic peptide antisera and use of the antibodies for localization and identification of ZP3 or ZPC domains of functional significance

The mammalian zona pellucida has an important function in the fertilization process. The zona pellucida protein 3 (ZP3 or ZPC) is the ligand for prima ry sperm binding and induces the acrosome reaction. In various species, ZP3 primary structures are highly conserved as revealed by cDNA cloning. The o bjective of these studies was to localize ZP3 protein using antisera genera ted against defined synthetic peptides that are specific for mouse or for h uman ZP3, Immunohistochemistry and transmission electron microscopy were ap plied to murine and human ovary sections. Immunochemical studies were perfo rmed in hemizonae pellucidae from microbisected human oocytes, Using the co mpetitive hemizona assay and various anti-ZP3 antibodies, we further intend ed to identify human ZP3 epitopes of functional significance. Our results s howed that antiserum AS ZP3-9 (mouse specific) detected mouse ZP3 protein i n mouse oocytes and in immunoblots, whereas AS ZP3-14 (human specific) dete cted human ZP3 protein in human ovary sections, native hemizonae pellucidae and in immunoblots, ZP3 material was also detected in cumulus cells by imm unohistochemistry. Ultrastructural studies showed an equal distribution of ZP3 throughout the zona pellucida. The human competitive hemizona assay rev ealed that none of the anti-ZP3 synthetic peptide antisera affected sperm b inding suggesting that those epitopes are not involved in primary sperm bin ding. Anti-porcine ZP3 beta protein antibodies (polyclonal) blocked human s perm-zona pellucida binding. In summary, these anti-ZP3 synthetic peptide a ntibodies specifically reacted with intact ZP3 protein (murine and human) b ut did not inhibit human sperm-zona pellucida binding; anti-ZP3 antibodies can therefore be used as biomarkers for ZP3 localization and function.