Recombinant Human Zona Pellucida Protein ZP3 Produced by Chinese Hamster Ovary Cells Induces the Human Sperm Acrosome Reaction and Promotes Sperm-Egg Fusion

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Abstract
Studies on the role of specific molecules in the human fertilization process and additional assessments of potential applications for these proteins are hampered by the limited amount of available biological material. However, this drawback might be circumvented by the recent cloning of several gamete-specific genes, which opens possibilities for the 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 constructed containing the natural third and fourth introns of the gene and a truncated intron between exons 2 and 3. This ZP3 DNA was transfected to Chinese hamster ovary cells, and a single-cell clone producing the recombinant ZP3 protein (recZP3) was generated. Western blot analysis of culture medium from these cells showed that recZP3 has a molecular mass +/- 5 kDa smaller than that of natural ZP3. Under reducing conditions, it migrates at an apparent molecular mass of 55-60 kDa. RecZP3 induced the sperm acrosome reaction and promoted fusion of human spermatozoa with zona-free hamster oocytes, indicating that the recombinant protein is biologically active. RecZP3 provides an attractive tool for studying the initial stage of the human fertilization process. Furthermore, it might have clinical applications in the development of diagnostic tests for male infertility and serve as target antigen in the design of contraceptive vaccines.

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