Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa

The human zona pellucida (ZP) is composed of three major glycoproteins: ZP1, ZP2, and ZP3. The aim of this study was to clarify the role of ZP2 by focusing on the polypeptide structure. We produced in Escherichia coli a recombinant human ZP2 protein (rec-hZP2) corresponding to amino acid sequence 1-206 of the mature protein. The final yield of rec-hZP2 protein was 80 microg/ml Luria Broth medium. After 2-h incubation of human spermatozoa with rec-hZP2 in vitro, an immunofluorescent study indicated that rec-hZP2 bound only to acrosome-reacted spermatozoa. The binding site migrated from the acrosome to the midpiece of the spermatozoa. Rabbit and mouse antisera produced against rec-hZP2 stained native human ZP in the immunofluorescent study, and significantly blocked human sperm binding and penetration into human ZP as compared to control values. The N-terminal polypeptide portion of human ZP2 was shown to contain a binding site for acrosome-reacted spermatozoa and to play an important role in secondary sperm binding and penetration into the ZP.