Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum

Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum. The functions of ODF have not yet been clearly elucidated. We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30-32-kDa polypeptide. This protein was analyzed by sequencing peptides derived following limited proteolysis. Peptide sequences were found to match VDAC2 and VDAC3. VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing. Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits. Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3. In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs. Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF. Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF. Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria

Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

International audienceVDAC (Voltage Dependent Anion selective Channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes three genes encode for VDAC. Nevertheless the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform characterized from living tissues until now. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS-PAGE two major and one-faint bands in the Mr range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie staining intensities. Western blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher Mw VDAC2 protein allowed the coverage of the whole protein with the exception of the tri-peptide A24AR26. In the same material it was revealed the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97). Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Step-wise increases in membrane conductance were observed with a predominant conductance of about 3.5 nS in 1 M KCl. Very often small short-lived fluctuations were observed with single-channel conductance of about 1.5 nS. Bovine spermatozoa VDAC2 was anion-selective and showed voltage dependence. This is the first work reporting the purification and characterization of VDAC2 from a mammalian tissue

Polyclonal VDAC3 antibody decreases human sperm motility: a novel approach to male contraception

<p><strong>Background:</strong> Voltage dependent anion channels (VDAC) mediate transport of anions, cations and ATP which play an important role in sperm motility. This study was aimed to examine the effect of polyclonal VDAC3 antiserum to human sperm motility.</p><p><strong>Methods:</strong> Polyclonal VDAC3 antiserum used in this study was produced in rabbits by immunization of VDAC3-specific synthetic peptides.  Preimmunserum was collected before immunization and used for control experiment. Recognition of VDAC3 antiserum to antigen in human sperm was performed by western blot. Thirty sperm samples obtained from fertile men which had high quality of sperm motility were washed and collected by Percoll gradient. Sperm motility was assessed by means of evaluation of sperm velocity (seconds per 0.1 mm distance) and the number of unmoved sperm (million per ml) which were observed 0 minute, 30 minutes and 60 minutes after addition of VDAC3 antiserum and preimmunserum as a control. Both data were analyzed by SPSS 13.0 software.</p><p><strong>Results:</strong> VDAC3 antiserum recognized VDAC3 protein in human sperm. Statistical analysis demonstrated that there were increasing numbers of unmoved spermatozoa after addition of anti-VDAC3 antiserum in vitro for 60 minutes observation compared with preimmunserum (control). We found also that sperm velocity decreased signifi cantly after giving anti-VDAC3 antiserum in vitro for 0 minute, 30 minutes, and 60 minutes compared with pre-immunee serum (control).</p><p><strong>Conclusion:</strong> VDAC3 antiserum can decrease motility of human sperm. and may provide a novel principle of male contraception in the future. <em><strong>(Med J Indones 2011; 20:5-10)</strong></em></p><p><strong>Keywords:</strong> <em>VDAC3 antiserum, sperm, motility, contraception</em></p