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.
VDACs (voltage-dependent anion channels) are a widespread class of pore-forming proteins (30–35 kDa) mainly found in the OMMs (outer mitochondrial membranes) of most eukaryotic organisms (for reviews, see [1,2]). In addition to mitochondrial localization, in the past few years several reports demonstrated the presence of VDACs in the plasma membrane and caveolae-like domains, sarcoplasmic/endoplasmic reticulum, nuclear envelope and endosomes (for reviews, see [3,4]).
VDACs have conserved sequences, and three different VDAC genes encoding distinctly expressed isoforms are known to exist in vertebrates [4,5]. Little is known in mammals about the tissue-specific distribution, intracellular localization and the specific role of each isoform [5–7]. The existing information derives mainly from recombinant proteins expressed in model organisms like yeast or mutant mice [5,8–10]. This paucity of information is due to the fact that, to date, mainly VDAC1 has been purified and characterized from different animal tissues, in contrast with VDAC2, which was until now only obtained from bovine spermatozoa .
The basic, known function of the VDAC is to form a diffusion pore with highly conserved characteristics in the OMM. The OMM functions as a link between mitochondrial metabolism and the rest of the cell. The involvement of VDAC in metabolic pathways and interactions with other supramolecular structures has been investigated [2,4]. The VDAC has been implicated to play a significant role in the programmed cell death pathway (for a review, see ). Other roles attributed to VDACs were the participation in calcium trafficking in the cell and the interaction with cytoskeletal proteins [4,13–15].
Currently, there is only scant information about the expression and function of VDAC proteins in gametes. In a previous study, we reported the presence of VDAC in the ODFs (outer dense fibres) of the bovine sperm flagellum  and their involvement in the regulation of essential sperm functions . Furthermore, we have obtained and characterized highly enriched VDAC2 from bovine spermatozoa .
Steinacker et al.  reported the localization of VDAC in the plasma membrane of Xenopus laevis oocytes. The expression and subcellular distributions of porin 1 and 2 (VDAC1 and 2) were also reported in the ovary of Drosophila melanogaster [19,20]. Wang et al.  also identified VDAC1 and 2 in a two-dimensional electrophoresis analysis of human ovary relating it to a possible role in the atresia of follicles.
At present, there is no evidence regarding the presence and the function of VDAC in mammalian oocytes. The present study provides the first report of the expression and localization of endogenous VDAC in immature and mature porcine oocytes as well as the purification of VDAC2 from porcine oocytes.
MATERIALS AND METHODS
Collection of oocytes and in vitro maturation
Ovaries of pre-pubertal gilts were transported at 37°C from a local abattoir in physiological saline solution containing 100 mg/ml kanamycin. Cumulus–oocyte complexes were aspirated from non-atretic follicles (3–6 mm diameter) and only oocytes with evenly granulated cytoplasm and several layers of cumulus cells were selected. The in vitro maturation was performed following the method described by Romar et al. . For all experiments, granulosa cell-free immature oocytes [GV (germinal vesicle)] and mature oocytes [MII (meiosis II)] were used.
RT–PCR (reverse transcription–PCR)
The total cellular RNA extracted from ten porcine oocytes using an RNeasy mini-kit (Qiagen, Hilden, Germany) was reversed-transcribed into the first-strand cDNA through Omniscript reverse transcriptase (Qiagen). The subsequent amplification of the desired genes was performed using Taq DNA polymerase (Eppendorf, Hamburg, Germany) according to the manufacturer's protocol. The primer sequences derived from porcine cDNA sequences (GenBank® Nucleotide Sequence Database) were constructed as follows: VDAC1, 5′-CAAAATCTGAGAATGGACTGGAA-3′ and 5′-TTGGTGAGAAGGATGAATCAAAG-3′; and VDAC2, 5′-AAATCAAAGCTGACAAGGAA-3′ and 5′-GACTTTTGCAGAAATGGAAG-3′. As a control for amplification, 5′- and 3′-primers for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used (5′-CCAGAAGACTGTGGATGGCC-3′ and 5′-CTGACGCCTGCTTCACCACC-3′). The amplification was conducted for 35 cycles at 94°C for 45 s, at an annealing temperature (57°C) for 45 s and at 72°C for 90 s with a final step of 72°C for 5 min.
Protein detection by immunoblotting
Oocytes were incubated at 70°C in a minimum volume of 0.9% NaCl for 1 h, then dissolved 1:1 (v/v) in oocyte sample buffer [5% (w/v) SDS, 100 mM Tris/HCl, pH 7, 25 mM EDTA, 12% glycerol, 2% Bromophenol Blue and 10% 2-mercaptoethanol] and vortex-mixed for 5 min followed by a last heating step at 95°C. SDS/PAGE was performed by the method of Laemmli  in 12% (w/v) polyacrylamide gels loaded with an extract from 300 oocytes per lane.
Proteins were subjected to immunoblotting following a previously published protocol . The antibodies used were the P1/6 AS (antiserum) (anti-VDAC1 antibody), monoclonal anti-human type I porin antibody (P31HL; Calbiochem, Darmstadt, Germany) and AS P2/45 (anti-VDAC2 antibody) at appropriate dilutions. Characterization and specificity of the antibodies has been previously described .
Purification and protein identification by MS of oocyte VDAC
Protein samples were prepared from immature oocytes (3300 cells) solubilized in Triton X-100 extraction buffer [2% (v/v) Triton X-100, 150 mM NaCl, 50 mM Tris/HCl, pH 7.4, 2 mM DTT (dithiothreitol), 1 mM EDTA, 10 mM benzamidine and 0.2 mM PMSF]. The suspension was incubated for 90 min at 4°C and, finally, centrifuged at 13800 g for 45 min. The supernatant (extract A) was stored at –80°C until it was used. The remaining sediment was subjected to a second solubilization treatment as described above (extract B). The proteins from extracts A and B were diluted 1:1 with 2% Triton X-100 extraction buffer. Each extract was applied to a dry hydroxyapatite/celite (2:1, w/w) column . The first 2 ml of the pass-through (VDAC-containing fraction) was collected separately in 1 ml fractions and stored at –20°C until it was used. Both extracts were subjected to semi-preparative SDS/PAGE (12% separating polyacrylamide gels) as described by Laemmli , and proteins were visualized by staining with SilverQuest (Invitrogen, Karlsruhe, Germany). Protein bands were excised and subsequently subjected to MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) analysis (ChromaTec, Greifswald, Germany). Peptide mass ‘fingerprinting’ database searching was carried out using software available online at http://www.matrixscience.com/.
Immunocytochemical localization of VDAC proteins
For immunofluorescence microscopy, porcine oocytes were fixed in 3.7% (w/v) paraformaldehyde (Sigma, Munich, Germany) in PBS (120 mM NaCl, 4 mM KH2PO4 and 10 mM Na2HPO4, pH 7.4) for 30 min at room temperature (20–25°C), washed three times for 5 min with PBS drops and blocked for 1 h with MP-PBS (PBS supplemented with 1% non-fat dried skimmed milk powder) (EuroClone, Milano, Italy). Incubation with primary antibody P31HL (anti-VDAC1 antibody; Calbiochem), AS P1/6 (anti-VDAC1 antibody) and anti-VDAC2 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was performed for 60 min followed by three washes in 0.1% MP-PBS. Secondary FITC-conjugated antibodies were applied for 30 min: anti-mouse IgG (Sigma) for P31HL antibodies; anti-rabbit IgG (Sigma) for AS P1/6; and anti-goat IgG (Santa Cruz Biotechnology) for anti-VDAC2 antibodies. During the last 5 min of incubation, propidium iodide (100 μg/ml; Sigma) was added to detect maturation stages. Finally, labelled oocytes were mounted on to slides, examined and recorded using a laser scanning confocal microscope (LSM 510; Zeiss Axioplan imaging, Goettingen, Germany). The microscope was equipped with an argon and helium–neon laser.
For the assessment of intracellular localization of VDAC in immature oocytes, the paraformaldehyde-fixed cells were permeabilized for 5 min in 0.1% Triton X-100 (Serva, Heidelberg, Germany) in PBS. The antibodies used were monoclonal anti-(human type I porin) antibody, P31HL (Calbiochem), AS P1/6 (anti-VDAC1 antibody), AS P2/45 (anti-VDAC2 antibody) and anti-VDAC2 antibody (Santa Cruz Biotechnology) at appropriate dilutions.
RT–PCR revealed that VDAC1 and VDAC2 mRNAs were expressed in porcine oocytes (Figure 1). Amplification of the coding sequences was detected in GV (VDAC1, lane A; VDAC2, lane B) and MII (VDAC1, lane C; VDAC2, lane D) stage oocytes. The primer pairs generated amplicons of 219 bp (VDAC1) and 231 bp (VDAC2) and the positive control GAPDH is indicated by an amplification product of 248 bp in GV and MII (GAPDH, lanes A–D). All amplicons were sequenced on both strands (MWG Biotech, Ebersberg, Germany) and confirmed by BLAST analysis.
RT–PCR of VDAC1 and VDAC2 isoform sequences in porcine mRNA obtained from GV and MII oocytes
Protein detection by immunoblotting
Figure 2 shows immunoblot results using protein extracts obtained from 300 GV and 300 MII stage oocytes. VDAC proteins detected with the AS P1/6 (anti-VDAC1 antibody, lanes 1 and 2) and AS P2/45 (anti-VDAC2 antibody, lanes 3 and 4) demonstrate the presence of the two VDAC isoforms, VDAC1 and VDAC2, in GV and MII oocyte protein extractions. A strong immunoreaction at the approximate molecular mass of 32 kDa was visible in GV (lane 1) and MII oocytes (lane 2) using anti-VDAC1 antibodies. In contrast, anti-VDAC2 immunodetection presented a different staining pattern between immature and mature oocytes. In immature cells, a polypeptide band of approx. 30 kDa was observed; however, a faint immune reaction with a 50–55 kDa peptide was also visible (lane 3). In mature oocytes, a single, strong immune reaction was observed at the molecular mass range of 50–55 kDa (lane 4), while the 30 kDa immunoreactive protein band was missing. In control experiments with the respective pre-immune sera, no immunoreactions were noted (results not shown).
Immunoblotting of VDAC1 and VDAC2 from porcine oocytes
Purification and protein identification by MS of oocyte VDAC
The purification procedure of porcine oocyte VDAC was adapted from the method described previously for mammalian VDAC protein . Proteins from immature porcine oocytes (n=3300) were isolated by a two-step sequential extraction (extracts A and B) as described in the Materials and methods section.
In extract A, no VDAC protein was detected (results not shown). Extract B contained a single faint protein band of 30–32 kDa in the first fraction (results not shown), while a greater amount was found in the second fraction (Figure 3). The protein band from fraction 2 was excised from the gel and subjected to MALDI–TOF-MS followed by peptide mass fingerprinting analyses. The number of peptides matched covered 31% of the VDAC2 (S. scrofa) protein.
Purification of VDAC2 protein from immature porcine oocytes
Immunocytochemical localization of VDAC proteins in porcine oocytes
In Figures 4(A)–(C), GV stage non-permeabilized oocytes were prepared and assayed with anti-VDAC antibodies. The anti-VDAC1 serum, namely AS P1/6, and the monoclonal anti-human type 1 VDAC antibodies, namely P31HL (Calbiochem), yielded a dense dot-like arrangement of fluorescent labelling primarily confined to the oocyte surface (Figures 4A and 4B). For the polyclonal antibody AS P1/6, additional staining of the zona pellucida was observed (Figure 4A, arrow). No distinct labelling of the oocyte surface was observed in control oocytes, which were treated only with the secondary antibody and the respective pre-immune serum or IgG2a (Figure 4C). VDAC2 antibodies (Santa Cruz Biotechnology) displayed no fluorescent labelling; however, some cells exhibited signs of lesions and disturbance of cell integrity (results not shown).
Immunolocalization of VDAC1 and VDAC2 proteins in porcine oocytes
For the assessment of intracellular VDAC polypeptides, membrane-permeabilized oocytes (GV and MII stages) were probed with anti-VDAC antibodies. Oocytes were completely scanned and photographed in three planes; however, mainly the equatorial plane data were included since it gave the most descriptive information.
As shown in Figure 4 for GV stage oocytes (D), the AS against VDAC1, namely AS P1/6 (D), presented a dense immunofluorescent labelling around the cortical area of the oocyte. Identical results were observed when immature oocytes were probed with another anti-VDAC1 antibody, P31HL (Calbiochem; results not shown). The assessment of MII porcine oocytes with both anti-VDAC1 antibodies yielded the same immunofluorescence pattern as that observed in GV stage porcine oocytes (results not shown).
By using anti-VDAC2 antibodies from Santa Cruz Biotechnology, immunolabelling was observed in most of the GV oocytes tested (Figure 4E). In these immunofluorescence-positive cells, variable spherical clusters, not evenly distributed throughout the cortical area, were detected (details observed in the oocyte cortical area of Figure 4F). However, when porcine MII oocytes were tested with anti-VDAC2 antibodies from Santa Cruz Biotechnology, no specific reaction was observed (results not shown). The anti-VDAC2 antibody (AS P2/45) displayed no specific fluorescent labelling in immature and mature porcine oocytes (results not shown).
In previous studies, we have reported the expression, localization and functional characterization of VDAC isoforms in bovine testis and spermatozoa [11,16,26]. Furthermore, we presented new results that indicate the participation of VDAC proteins in essential sperm functions . In contrast, VDACs in mammalian oocytes have not yet been investigated.
Using RT–PCR, we demonstrated for the first time the expression of VDAC1 and VDAC2 within immature (GV) and in vitromatured (MII) porcine oocytes. The amplification products detected in porcine oocytes corresponded to the published coding sequence. The presence of additional sequences corresponding to an unspliced variant, as has been described previously for porin 2 (VDAC2) in D. melanogaster, remains to be elucidated .
Specific antibodies generated against VDAC1 and VDAC2 allowed investigation of the expression pattern of VDAC proteins in oocytes. Immunoblot analyses demonstrated the expression of VDAC1 in GV and MII maturation stages. Testing of immature porcine oocytes with anti-VDAC2 antibodies yielded VDAC2 protein of same molecular mass range of approx. 30–32 kDa as has been reported previously for sperm VDAC2 . Additionally, we noticed a faint immunoreactive band with a higher molecular mass of approx. 50–55 kDa. Interestingly, in mature oocytes (MII), only a single, strong immunoreaction with anti-VDAC2 antibodies was observed with the 50–55 kDa protein band, while the original immunoreactive 30–32 kDa VDAC protein disappeared. This result supports the hypothesis of the expression of two VDAC2 sub-isoforms, with different polypeptide lengths, and with a pattern of similar post-translational modifications . Guarino et al.  suggested that a high-molecular-mass VDAC protein in Drosophila ovaries could be the product of mRNA still containing intron sequences.
We sought to further investigate the identity of the immunoreactive protein bands by purifying porcine oocyte VDAC proteins to apparent homogeneity and subsequent sequencing of the polypeptides using hydroxyapatite/celite purification  and MALDI–TOF-MS analyses respectively. To obtain sufficient material for sequencing, high amounts of granulosa cell-free oocytes and a two-step protein extraction method were necessary. Here we report, to our knowledge for the first time, the purification of the 30–32 kDa VDAC2 protein from mammalian immature oocytes.
VDAC1 and the VDAC2-related 50–55 kDa polypeptide, identified by immunological methods in immature porcine oocytes, could not be detected in the VDAC protein-containing hydroxyapatite/celite throughput. Presumably, the amount of these proteins was too low or the proteins were retained in the hydroxyapatite/celite column .
Having identified VDAC proteins in mature and immature porcine oocytes, the subcellular distribution of these proteins was studied. The cytochemical analyses of non-permeabilized oocytes with anti-VDAC1 antibodies revealed the presence of VDAC1 in the plasma membrane region. Very similar staining patterns were obtained with the two different anti-VDAC1 antibodies. However, AS P1/6 additionally displayed immunofluorescence of the zona pellucida. Compared with the specific staining pattern of the monoclonal anti-VDAC1 antibody P31HL (Calbiochem), this observation is most probably due to non-specific binding to this extracellular oocyte matrix protein.
The fluorescence pattern of anti-VDAC1 antibodies is reminiscent of the cytological staining observed in X. laevis oocytes . Furthermore, the dot-like labelling pattern at the cell surface suggests a plasma membrane localization for VDAC1. Because the oocytes assayed in our present study were not permeabilized, we assume that the VDAC1 epitopes detected by the two antibodies are localized at the extracellular plasma membrane domain of the oocyte. The first evidence for an extra-mitochondrial localization of VDAC protein in the plasma membrane of human B lymphocytes was published in 1989 . Several reports on the presence of VDAC as an integral membrane protein of various kinds of cells and organelles supported this study [2,3,29–31]. Our results confirm the localization of VDAC1 in the plasma membrane of porcine oocytes; however, no information on the function of plasma membrane-bound VDAC1 in oocytes is yet available. Several studies suggest that VDAC in the plasma membrane of somatic cell types acts as an anion channel involved in volume regulatory mechanisms [32,33]. The participation of VDAC1 in the volume regulatory process of mammalian spermatozoa was reported recently for gametes . VDAC2 was not observed in the oocyte plasma membrane, suggesting separate localizations and functions of both isoforms in the oocyte.
After having found that anti-VDAC1 antibodies bind to the outer surface of the oocyte plasma membrane, we were interested in identifying VDAC proteins in the ooplasm of immature and mature porcine oocytes. Using confocal microscopy, it was possible to detect VDAC1 and VDAC2 proteins in different localizations of the porcine ooplasm. VDAC1 was clearly observed at the cortical area of immature and mature oocytes. Thus the distribution of VDAC1 proteins seemed to be stable throughout meiotic maturation. In the literature, it has been reported that in the GV stage of mammalian oocytes, actin filaments are distributed to form a relatively thick, uniform area around the cell cortex and are also found near the GV. After GV breakdown, microfilaments are predominantly found at the cortex of the cell and around the female chromatin . The interaction of VDAC with other cellular proteins, especially those of the cytoskeleton, has been reported previously [15,35]. G-actin has been demonstrated to modulate VDAC gating in Neurospora crassa , whereas in HeLa cells the dynein light-chain Tctex1 was shown to regulate VDAC . Furthermore, VDACs were identified as binding sites for MAP2 (microtubule-associated protein 2) in the outer membrane of rat brain mitochondria [15,36]. The localization of VDAC1 at the cortical area of the oocyte might indicate an interaction of VDAC1 with cytoskeleton proteins that are predominantly found in this area of the cell.
The observed pattern of VDAC1 distribution could also suggest a mitochondrial localization. The presence of VDAC protein on the outer membrane of mitochondria isolated from eukaryotic cells is well established [37,38]. During porcine oocyte maturation, the distribution of oocyte mitochondria was reported in the perinuclear area in GV stage oocytes, and large mitochondrial foci form and relocate to the inner cytoplasm in mature oocytes . In mouse oocytes, mitochondria are clustered at the periphery of the large nucleus and the cortical area, but they disperse throughout the cytoplasm during maturation. This pattern of distribution may be related to the high energy requirement in the cortex, as the oocyte requires the support of cumulus cells at this stage .
Staining patterns observed with anti-VDAC2 antibodies differed from that of the VDAC1 isoform. In immature porcine oocytes, VDAC2 antigen was detected as distinct but not evenly distributed spherical clusters throughout the cortical area of the ooplasm. Thus it can be speculated that VDAC1 and VDAC2 are present in different intracellular compartments of the oocyte. Our results implicate that, in oocytes, VDAC subtypes might be involved in the regulation of different cell functions (e.g. cell maturation or apoptosis). However, the exact localization and role of VDAC in the regulation of particular oocyte functions remain to be investigated.
In mature oocytes, the labelling pattern of VDAC2 protein completely disappeared after in vitro maturation, apparently indicating an interrelation of VDAC2 distribution or integrity with the developmental stage of the cell. Nevertheless, the lack of immunoreactivity observed in mature oocytes could also be explained by a post-translational change of the conformation of VDAC2 protein that hinders binding of the antibody to the native protein. It has been shown previously that some of the oocyte organelles exhibit dramatic changes during meiotic progression. Although VDACs were originally thought to be located only in the OMM, several investigations reported the presence in other cell compartments, e.g. nuclear envelope, endosomes, sarcoplasmic/endoplasmic reticulum, caveolae and other organelles of somatic cells [3,4,40–42]. The distribution of caveolae in Caenorhabditis elegans oocytes  resembles that observed in our immunofluorescence studies with anti-VDAC2 antibody. However, preliminary double staining experiments, containing anti-VDAC2 and anti-caveolin 1 antibodies, showed only a partial overlapping of both proteins. More research is essential to determine VDAC localization in these or other organelles in mammalian oocytes.
Anti-VDAC2 antibodies clearly exhibited different labelling patterns within immature oocytes. In the future, it will be of great interest to investigate whether the difference observed in VDAC2 labelling is particular to the individual characteristics of each oocyte (e.g. oocyte quality). The identification of quality markers for oocyte fertilization capacity would be of great help in clinical applications and in vitro production.
The data obtained for VDAC2 using immunofluorescence are in line with the immunoblot experiments performed in the present study, where shifts of apparent molecular masses of VDAC antigens during maturation were detected. The immunoreactive band at 30–32 kDa was no longer found after oocyte maturation, and a strong immunoreaction was observed in the 50–55 kDa molecular mass range. The 50–55 kDa VDAC antigen, which is evidently the only VDAC2-related protein in the ooplasm of mature oocytes, could possibly represent a homodimerization/oligomerization product of these integral membrane proteins [44–46] or a tight complex of VDAC with other polypeptides . However, it is tempting to speculate that either alterations of intracellular distribution of VDAC2 or protein–protein interactions of VDAC isoforms are causally related to the shift of apparent molecular masses during the maturation of oocytes.
The identification and localization of VDAC isoforms in different compartments of maturing porcine oocytes unveiled in the present study could be the basis for better understanding of the molecular mechanisms of oocyte functions as well as the basis for novel clues concerning the role of VDAC proteins in the regulation of physiological processes of cells.
matrix-assisted laser-desorption ionization–time-of-flight MS
PBS supplemented with 1% non-fat dried skimmed milk powder
outer mitochondrial membrane
voltage-dependent anion channel
We thank the staff of the Institute for Animal Breeding Biotechnology at the FAL (Federal Agricultural Research Centre; Neustadt, Germany) for their excellent technical assistance and support.
This work was supported by the Deutsche Forschungsgemeinschaft [grant number GRK 533]. This paper includes parts of the thesis of M.C.C.