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 primary sperm binding and induces the acrosome reaction. In various species, ZP3 primary structures are highly conserved as revealed by cDNA cloning. The objective of these studies was to localize ZP3 protein using antisera generated against defined synthetic peptides that are specific for mouse or for human ZP3. Immunohistochemistry and transmission electron microscopy were applied to murine and human ovary sections. Immunochemical studies were performed in hemizonae pellucidae from microbisected human oocytes. Using the competitive hemizona assay and various anti-ZP3 antibodies, we further intended to identify human ZP3 epitopes of functional significance. Our results showed that antiserum AS ZP3-9 (mouse specific) detected mouse ZP3 protein in mouse oocytes and in immunoblots, whereas AS ZP3-14 (human specific) detected human ZP3 protein in human ovary sections, native hemizonae pellucidae and in immunoblots. ZP3 material was also detected in cumulus cells by immunohistochemistry. Ultrastructural studies showed an equal distribution of ZP3 throughout the zona pellucida. The human competitive hemizona assay revealed that none of the anti-ZP3 synthetic peptide antisera affected sperm binding suggesting that those epitopes are not involved in primary sperm binding. Anti-porcine ZP3β protein antibodies (polyclonal) blocked human sperm–zona pellucida binding. In summary, these anti-ZP3 synthetic peptide antibodies specifically reacted with intact ZP3 protein (murine and human) but did not inhibit human sperm–zona pellucida binding; anti-ZP3 antibodies can therefore be used as biomarkers for ZP3 localization and function.