Molecular Cloning Of IZUMO3CDNA Of The Cashmere Goat And Sheep And Localization In Testes And Sperm

Mammalian fertilization consists of an intricate cascade of cell-cell interactions that culminate in the fusion of sperm and egg membranes and formation of a zygote. Multiple molecules had been identified in sperm and egg to be involved in sperm-egg fusion, however, the mechanisms of interaction among those proteins by which acrosome-reacted spermatozoa bind to and fuse with the oolemma remain unknown. Among these candidate molecules for sperm-egg fusion, the Izumo1had been found to be essential for the sperm-egg fusion in knockout mouse. Recently Izumo1was found to be a member of the Izumo family because it has the Izumo domain with polymerization potential in its N-terminal region. Other three Izumo family members are Izumo2, Izumo3and Izumo4, all having the same structural domain and being expressed in testicle with their functions unknown. Our lab has reported molecular cloning and characterization of Izumol gene and cDNA in cashmere goat and sheep. In the present study we cloned the Izumo3cDNA in sheep and cashmere goat. The cloned Izumo3are of946bp, with98.3%identity between cashmere goat and sheep, and97.3%with cattle Izumo3. Results of bioinformatics analysis indicated that Izumo3is a transmembrane protein of240aa, with a putative signal peptide of22aa at its N terminus and a transmembrane domain (179-201aa).We inserted the cDNA coding the mature peptide into prokaryotic expression vector pET-HIS and expressed recombinant HIS-Izumo3protein in E. coli. Following purification and collection, we immunized mouse with the recombinant protein to generate anti-Izumo3ascites polyclonal antibodies. The ascites polyclonal antibodies were purified by glutaraldehyde cross-linking method and were found to recognize the Izumo3in the total protein extract of testis specifically. To demonstrate the expression pattern of Izumo3in testis and its localization in sperms, we carried out immunohistochemistry assay on the section of cashmere goat testes and immunocytochemistry assay on sperm cells. The results showed that Izumo3protein exists in the cells in all meiosis stages. It locates in the posterior half region of the intact sperm head behind the equator and the tail. This position pattern does not change after the acrosomal reaction.