Identification of mouse ZP3 protein in mammalian oocytes with antisera against synthetic ZP3 peptides.

Abstract

The mouse zona pellucida (ZP) protein ZP3 plays an important role in the process of fertilization by mediating sperm binding and the acrosome reaction. ZP3 primary structures are highly conserved, as revealed by cDNA cloning. We raised antisera against synthetic peptides that are either conserved in the structure of ZP3 from different mammalian species (AS ZP3-5 and AS ZP3-6) or specific for mouse ZP3 (AS ZP3-2). In ovary sections, AS ZP3-2 revealed immunoreactivity only to mouse ZP. AS ZP3-5 and AS ZP3-6 reacted with mouse, human, rat, hamster, porcine, and bovine ZP proteins. In porcine oocytes, immunoreactive material was highly abundant in the ooplasm. Immunoblots showed that antiserum AS ZP3-5 recognized the mouse ZP3 protein. In porcine ZP preparations, AS ZP3-5 recognized a 53-kDa ZP protein. No reaction was observed with purified porcine ZP3 alpha or with ZP3 beta. Immunofluorescence studies revealed that AS ZP3-5 and AS ZP3-6 antibodies react with ZP of isolated porcine and human oocytes. Our results show that antisera against synthetic mouse ZP3 peptides can be used as markers for the identification of ZP3-like proteins in mammalian oocytes and might be useful tools for the evaluation of ZP integrity and ZP3 function.