Abstract
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.
Highlights
Eukaryotic voltage-dependent anion channels (VDAC)1 or porins are small pore-forming proteins first identified in outer mitochondrial membranes [1]
We reported the sequencing of the VDAC2 cDNA from bovine testis and demonstrated a high identity to the murine, rabbit, and human subtypes at both the nucleotide and amino acid levels [15]. mRNA analysis revealed the expression of VDAC2 in the bovine testis; high levels of VDAC2 proteins were found in spermatocytes, spermatids, and spermatozoa
By limited proteolysis of this protein band excised from the gel, we identified amino acid sequences matching a VDAC/porin cDNA present in the public databases
Summary
5Ј-AAA GGA TTC ATG GCT GTG CCA CCC ACG TAT 5Ј-AAA TAG GTC TTA TGC TTG AAA TTC CAG TCC 5Ј-TAG GAG CTC ATG TGT ATT CCT CCA TCA TAT 5Ј-TAG GTC GAC TTA AGC CTC CAA CTC CAG 5Ј-TAG GAG CTC ATG TGT AAC ACA CCA ACG TA 5Ј-TAG GTC GAC TTA AGC TTC CAG TTC AAA TCC of human lymphocytes. We reported the sequencing of the VDAC2 (porin-2) cDNA from bovine testis and demonstrated a high identity to the murine, rabbit, and human subtypes at both the nucleotide and amino acid levels [15]. MRNA analysis revealed the expression of VDAC2 in the bovine testis; high levels of VDAC2 proteins were found in spermatocytes, spermatids, and spermatozoa. The ODF have been suggested to maintain the passive elastic structure and elastic recoil of the sperm flagellum needed for flagellar bending and possibly to protect it from shearing forces during epididymal transport [27]. We found evidence that high amounts of VDAC2 and VDAC3 are present in the ODF of the bovine sperm flagellum. The possible role of such pore-forming VDAC proteins in sperm ODF, which represents a non-membranous structure in mammalian spermatozoa, will be discussed
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