Human Sperm In The Voltage-dependent Anion Channel Protein In Basic And Clinical Research

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 that VDAC might play important roles in spermatogenesis, motility, capacitation and acrosome reaction. However, it remains unclear about the exact functions and molecular mechanisms 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 study the construction features and localization of VDAC in cellular ultrastructure, and for researching the possible interaction between VDAC and other proteins on the surface of human spermatozoa; discovering the possible roles and mechanisms of VDAC during sperm capacitation and acrosome reaction for initially exploring the functions of VDAC in human spermatozoa; studying the relationship between VDAC and asthenozoospermia for exploring the effect of VDAC on sperm motility from another point view and finding the clinical value of studying VDAC in human spermatozoa.Part I: the presence, expression and localization of VDAC in mature human spermatozoaSpecific 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. 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 neck. 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. Our study elucidates that VDAC protein is synthesized during spermatogenesis and is eventually localized to the plasma membrane of flagellar midpiece and neck of mature human spermatozoa.Part II the relationship between VDAC and mature human sperm motility and acrosome reactionMature human spermatozoa with normal motility were separated using a discontinuous Percoll gradient centrifugation, co-incubated with anti-VDAC antibody and cultured in vitro. The motion parameters reflecting sperm motility, cytoplastic free calcium (Ca²⁺) concentration, ATP content and acrosome reaction rate were detected to search for the possible influence of antibody co-incubation. We found that the co-incubation of anti-VDAC antibody with mature human spermatozoa led to reduced sperm motility and decreased cytoplastic Ca²⁺ concentration. However, cytoplastic ATP content and acrosome reaction rate did not show significant difference. Our study elucidates that the co-incubation can disturb the mediation of VDAC on Ca²⁺ transmembrane transport and exchange, cut down cytoplastic Ca²⁺ concentration, and finally leed to impaired sperm motility. However, decreased cytoplastic Ca²⁺ concentration does not inhibit normal acrosome reaction.Part III the relationship between VDAC and anthenospermiaHigh motile and low motile spermatozoa were separated respectively from normal semen of health donors and abnormal semen of patients with idiopathic asthenozoospermia using a discontinuous Percoll gradient centrifugation. Real-Time PCR was performed to detect mRNA abundance and difference of three VDAC subtypes between two groups with different sperm motility. We found that three VDAC mRNAs were present in mature human spermatozoa. The VDAC2 mRNA level in low motile spermatozoa was significantly higher than that in high motile spermatozoa. No significant differences of VDAC1 and VDAC3 mRNA levels were found between two groups. Our study elucidates that the high abundance of VDAC2 mRNA seems to have a positive correlation with low sperm motility. The abnormal gene transcription and protein expression of VDAC, especially VDAC2, may be one of the causes of some male infertility with idiopathic asthenozoospermia.