RECOMBINANT HUMAN ZONA-PELLUCIDA PROTEIN ZP3 PRODUCED BY CHINESE-HAMSTER OVARY CELLS INDUCES THE HUMAN SPERM ACROSOME REACTION AND PROMOTESSPERM-EGG FUSION

Studies on the role of specific molecules in the human fertilization p rocess and additional assessments of potential applications for these proteins are hampered by the limited amount of available biological ma terial. However, this drawback might be circumvented by the recent clo ning of several gamete-specific genes, which opens possibilities for t he production of recombinant proteins. By use of cDNA and genomic DNA fragments of the human ZP3 gene, which encodes a major constituent of the zona pellucida surrounding the oocyte, a 2.7-kb minigene was const ructed containing the natural third and fourth introns of the gene and a truncated intron between exons 2 and 3. This ZP3 DNA was transfecte d to Chinese hamster ovary cells, and a single-cell clone producing th e recombinant ZP3 protein (recZP3) was generated. Western blot analysi s of culture medium from these cells showed that recZP3 has a molecula r mass +/- 5 kDa smaller than that of natural ZP3. Under reducing cond itions, it migrates at an apparent molecular mass of 55-60 kDa. RecZP3 induced the sperm acrosome reaction and promoted fusion of human sper matozoa with zona-free hamster oocytes, Indicating that the recombinan t protein is biologically active. RecZP3 provides an attractive tool f ar studying the initial stage of the human fertilization process. furt hermore, it might have clinical applications in the development of dia gnostic tests for male infertility and serve as target antigen in the design of contraceptive vaccines.