PL 34. Voltage-dependent anion channels (VDAC) in human spermatozoa
The researcher in our lab found the amino acid sequence of voltage-dependent anion channels (VDAC) in human spermatozoa in the early study about the proteins on the surface of spermatozoa. A few studies now available about VDAC in mammalian reproductive tissues and cells have suggested VDAC might play important roles in spermatogenesis, motility control, capacitation and acrosome reaction. However, it remains unclear about the exact functions and molecular mechanism of VDAC in mammalian spermatozoa. Up to now, there are not any reports on the existence and function of VDAC in human spermatozoa. The aim of our study is as following: systematically exploring the presence, expression and localization of VDAC in human mature spermatozoa for further functional research; acquiring recombinant three VDAC proteins for producing specific monoclonal antibody to further study the functions of VDAC; discovering the possible roles and mechanism of VDAC during sperm acrosome reaction for initially exploring the functions of VDAC in human spermatozoa.
Specific primers coding three VDAC were designed and VDAC gene sequences were amplificated via PCR from human testis cDNA library. Proteins extracted from mature human spermatozoa were analyzed by western blot. The cellular localization of VDAC in mature human spermatozoa was detected using immunofluorescence. The changes in acrosomal integrity rate and intracellular Ca2+ concentration of human spermatozoa with or without VDAC antibody incubation during acrosome reaction were investigated respectively using FITCPSA staining and Fluo-3 AM.
Three recombinant VDAC proteins were produced through gene cloning and prokaryotic expression. We obtained three PCR productions of VDAC coding genes respectively from human testis cDNA library. We found that native VDAC protein existed in mature human spermatozoa mainly as the form of membrane protein. The native VDAC protein was localized to the midpiece of sperm flagella and necks. Three cloned plasmids containing VDAC open genes were acquired and the corresponding three recombinant proteins (34-40kDa) were produced mainly as the form of inclusion body in prokaryotic expression system, which would be beneficial to the large-scale recombinant protein production in our next research. The co-incubation of human spermatozoa with VDAC antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase.
In the current study, we systematically researched the presence, expression, localization and functions of VDAC in human mature spermatozoa for the first time. Our study elucidated that VDAC protein was synthesized during spermatogenesis and eventually localized to the plasma membrane of flagellar midpiece and neck of mature human spermatozoa. Furthermore, our study exhibits the functional role of VDAC on A23187-induced intracellular Ca2+ increase during acrosome reaction, which suggested that VDAC played putative roles in sperm functions through mediating Ca2+ transmembrane transport.
