Characterization of Voltage Dependent Anion Channel (VDAC) subtypes in mammalian follicles and potential physiological relevance

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CassaraMariaCarolina-2007-05-11.pdf (2.57 MB)

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2007

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http://dx.doi.org/10.22029/jlupub-13182

Abstract

This thesis comprises the study of the expression of VDAC subtypes in porcine and bovine oocytes in GV and MII stage oocytes by means of RT-PCR. The results demonstrate that VDAC1, 2 and 3 subtypes are expressed within oocytes of both species. Subsequently, quantitative expression of VDAC1, 2 and 3 mRNA transcripts in bovine oocytes from follicles with three different diameters was determined (group 1 <3mm; group 2, 4-6mm and group 3, 8-11mm) by real-time RT-PCR. A distinct difference in the relative expression was observed only for VDAC1 which presented down-regulation of expression with a magnitude factor of 2.4 in group 3 compared to group 1. Immunoblot studies using subtype specific anti-VDAC antibodies and porcine spermatozoa indicated the presence of VDAC1, 2 and 3 in male germ cells. Further characterization of VDAC polypeptides by MALDI-TOF MS and PMF analyses of 2D-gels from hydroxyapatite-celite purified VDAC from porcine spermatozoa protein confirmed the detection of VDAC2. Functional experiments in artificial membranes showed that biochemically isolated VDAC was successfully renatured by demonstrating voltage dependent electrophysiological channel properties and a single conductance of 1.6nS. In porcine oocytes VDAC1 and VDAC2 protein expression could be demonstrated by immunoblot experiments assaying VDAC proteins that derived from GV and MII stage oocytes. A shift of intensity of protein stain as well as differences in apparent molecular masses were observed with anti-VDAC2 antibody between GV and MII stage oocytes. MALDI-TOF MS and PMF analyses confirmed the presence of VDAC2 protein in GV stage oocytes. Using specific antisera against synthetic peptide sequences from VDAC subtypes immunohistochemical data suggest the presence of VDAC 1 and 2 in ooplasm of porcine oocytes as well as in the follicle cells with no clear change of localization during folliculogenesis. Subcellular localization of VDAC1 by confocal laser microscopy exhibited a dense arrangement of particular dotted fluorescent labeling over the entire oocyte surface suggesting the presence of VDAC1 in the porcine oocyte plasma membrane. Data obtained from permeabilized oocytes assayed with anti-VDAC1 and anti-VDAC3 antibodies show a clear pattern of spotlike immunofluorescent labeling localized particularly around the cortical area in GV and MII stage oocytes. Anti-VDAC2 immunostaining yielded ring-like clusters structures distributed on the cortical area in some but not in all GV stage oocytes. Microinjection of matured porcine and bovine oocytes with VDAC protein purified from porcine spermatozoa was performed in order to detect possible oocyte activation and/or calcium oscillation activity. The results showed no clear change of oocyte activity or calcium oscillation after intraooplasmic injection of purified sperm VDAC protein. Finally, blocking VDAC1 activity in in vitro maturating bovine oocytes using anti-VDAC1 antibodies resulted in a significant interruption of physiological maturation events. These results obtained in this study are the first that show particular biochemical characteristics, distributions and possible functions of VDAC subtypes during maturation and fertilization of porcine and bovine oocytes.

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