Anti‐ZP3 Antibodies Binding to the Human Zona Pellucida: Effect of Oocyte‐Storage Conditions

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions. METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm-ZP binding capacity under different HZA conditions. RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4 degrees C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt-stored, or frozen with 1,2 propanediol. CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol-treatment while the glycoprotein-receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.