VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. 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 bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue 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 molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

Sperm motility, regulation of cell volume, sperm capacitation, acrosome reaction and tight binding of spermatozoa to the zona pellucida are crucial events in the process of fertilisation. Voltage-dependent anion channels (VDAC) are highly conserved pore-forming proteins implicated in apoptosis, metabolite transport between mitochondria and cytosol, energy metabolism, and cell volume regulation in somatic cells. Several studies have demonstrated the presence of VDAC in cell compartments other than mitochondria. In previous studies using immunofluorescence, we were able to localise VDAC2 and VDAC3 in outer dense fibres of the bovine sperm flagellum. Furthermore, we described the presence of VDAC2 in the head of bovine sperm. In the present study, we confirm the localisation of VDAC2 in the acrosomal region of bovine spermatozoa using immunoelectron microscopy. After incubation with anti-VDAC antibodies raised against each VDAC isoform, bovine spermatozoa showed an increased loss of the acrosomal cap, noticeable changes in the surface of the head, coiled tails and an increased cell volume. The incubation of bovine spermatozoa with anti-VDAC antibodies might lead to alteration of the intracellular ion concentration that causes changes in the cell volume, followed by destabilization of the cytoskeleton and, finally, to loss of the acrosomal cap.

Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

The eukaryotic VDAC (voltage-dependent anion channel) is a pore-forming protein originally discovered in the outer membrane of mitochondria. It has been established as a key player in mitochondrial metabolism and ion signalling. In addition, in recent years, it has also been proposed that VDAC is present in extra-mitochondrial membranes, and it has been related to cytoskeletal structures. However, little is known about the presence and intracellular localization of VDAC subtypes in mammalian gametes. In the present study, we confirm the synthesis of VDAC1 and 2 subtypes in GV (germinal vesicle) and MII (meiosis II) stage porcine oocytes as well as their protein expression. A shift in the abundance of immunoreactive 32 kDa VDAC protein between GV and MII stage oocytes was observed with anti-VDAC2 antibody. Furthermore, subcellular localization by confocal laser microscopy demonstrated fluorescent labelling of VDAC1 over the entire oocyte surface, suggesting the presence of VDAC1 in the porcine oocyte plasma membrane and around the cortical area. Anti-VDAC2 immunostaining yielded ring-like clusters of structures distributed on the cortical area in some GV, but not in MII, stage oocytes. These results are the first data obtained for VDAC in mammalian female gametes and provide the basis for studying protein-protein interactions, distribution and possible functions of VDAC subtypes during maturation and fertilization of mammalian oocytes.