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) g lycoprotein crucially involved in fertilization. ZP3 plays a major rol e in sperm binding and induction of the acrosome reaction. In differen t species, ZP3 proteins differ in their primary structure as derived f rom cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertil ization outcome under in vitro conditions. METHOD: In these studies, w e used antisera generated against synthetic ZP3 peptides to compare an tibody binding patterns to ZP with sperm-ZP binding capacity under dif ferent HZA conditions. RESULTS: Analysis of antibody binding to hemizo nae derived from metaphase II human oocytes that were used either afte r refrigeration at 4 degrees C or stored in a hyperosmotic salt soluti on revealed a strong reaction with human ZP3. However, treatment of hu man oocytes using a protocol to freeze embryos with the addition of 1, 2 propanediol drastically reduced binding of ZP3 antibodies to the hem izonae. Nevertheless, no significant difference of sperm binding occur red 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-treatmen t while the glycoprotein-receptor remains intact. We conclude that ant isera 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.