Evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides

The mouse zona pellucida protein ZP2 plays an important role in the process of fertilization by mediating secondary sperm binding to mammalian oocytes . ZP2 primary structures are highly conserved as revealed by cDNA cloning. The aim of the study was to identify ZP2 domains of functional relevance. A ntisera were raised against synthetic peptides that are either conserved in the structure of ZP2 from different mammalian species (AS ZP2-20) or prese nt in the human ZP2 but not in the mouse ZP2 amino acid sequence (AS ZP2-26 ). Antibody binding to zona pellucida proteins was assessed by assaying the antisera with human hemizonae. Using human zonae pellucidae, we demonstrat ed that anti-ZP2 common antibodies and anti-ZP2 human peptide antibodies re act with human zona pellucida antigens. For the first time, ZP2 domains of functional relevance for human sperm-oocyte interaction could be identified applying the competitive hemizona assay. Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae, whereas treatment of h emizonae with AS ZP2-26 did not influence sperm-oocyte interaction. These r esults show that antibodies against synthetic ZP2 peptides react with ZP2 p rotein and that AS ZP2-20 identifies a linear ZP2 epitope that is of possib le functional importance for sperm-oocyte interaction.