Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes

The ZP2 protein is a zona pellucida glycoprotein that plays a major role in fertilization. It mediates secondary binding of spermatozoa and is one of the proteins that are involved in zona 'hardening'. ZP2 proteins were identified in various mammalian zonae pellucidae. Their primary structures are highly conserved as revealed by cDNA cloning. Antisera were used against synthetic peptides generated either against a ZP2 amino acid that is homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or antibodies against a synthetic human ZP2 peptide (AS ZP2-26). Immunoblots showed that antiserum AS ZP2-20 and AS ZP2-26 strongly recognized human ZP2 protein with an apparent molecular mass of about 72 kDa; both antisera reacted with a minor immunoreactive polypeptide at 96 kDa. In human ovary sections, both antisera revealed immunoreactivity to human zonae pellucidae. Immuno-electron microscopy demonstrated an equal distribution of ZP2 throughout the human zona pellucida. Considerable amounts of immunoreactive material were observed in the ooplasm; some ramification-like extensions of zona pellucida antigen were found close to cells surrounding the oocyte. Our results indicate that antisera against synthetic ZP2 peptides can be used as specific markers for the identification of ZP2 protein in human oocytes.