Bioscience Reports (2010) 30, 193-200 - M. C. Cassara and others

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Bioscience Reports (2009) 30, (193–200) (Printed in Great Britain)

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Original Paper

Voltage-dependent anion channels 1 and 2 are expressed in porcine oocytes

María Carolina Cassará*¹, Viviana Andrea Menzel†, Klaus‑Dieter Hinsch‡, Christine Wrenzycki§ and Elvira Hinsch*

*Department of Urology, Pediatric Urology and Andrology, Justus Liebig University of Giessen, Rudolf-Buchheim-Strasse 7, Germany, †Center of Dermatology and Andrology, Justus Liebig University of Giessen, Gaffkystrasse 14, D-35392 Giessen, Germany, ‡Borkum Riff Rehabilitation Clinic of the Federal Insurance for Salaried Employees' Institution (DRV-Bund), 26757 Borkum, Germany, and §University of Veterinary Medicine Hannover, Clinic for Cattle, Reproductive Medicine Unit, Bischofsholer Damm 15, 30173 Hannover, Germany

Key words: immunoblotting, indirect immunofluorescence, oocyte, porcine oocyte, reverse transcription–PCR (RT–PCR), voltage-dependent anion channel (VDAC).

Abbreviations used: AS, antiserum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GV, germinal vesicle; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MII, meiosis II; MP-PBS, PBS supplemented with 1% non-fat dried skimmed milk powder; OMM, outer mitochondrial membrane; RT–PCR, reverse transcription–PCR; VDAC, voltage-dependent anion channel.

¹To whom correspondence should be addressed (email carolina.cassara@yahoo.com).

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.