Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull.

AIM: To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. METHODS: The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA)/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline (CTC) assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. RESULTS: Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. CONCLUSION: 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.

[Immunogenicity of recombinant human zona pellucida-3 peptides expressed in E. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding].

The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides (r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348)) expressed in E. coli through immunizing rabbits, and to evaluate the efficacy of their polyclonal antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding respectively. Male New Zealand rabbits were immunized using r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) as antigen respectively, which was purified through an improved method of preparative gel polyacryulamide gel electrophoresis. The antibody response level of r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) immunogen in rabbits was determined by ELISA using mouse ZP3-5 (amino acid sequence(137 approximately 150) being completely conserved with huZP3(138 approximately 151) sequence) and specific huZP3-14 (amino acid sequence(327 approximately 340)) synthetic peptides as coating antigens respectively. The immunoreactivity and specificity of the anti-r-huZP3a(22 approximately 176) and anti-r-huZP3b(177 approximately 348) antisera with each r-huZP3 peptides, were tested by immunoblot and immunohistochemistry (using native huZP and human ovary section) respectively. A competitive hemizona assay (HZA) was used to evaluate the efficacy of the antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding. Both r-huZP3 peptides were able to induce higher antibody titers in rabbits. Each antiserum could specifically recognize or bind to each target r-huZP3 peptide expressed in E. coli and native human ZP in vitro. The antisera also inhibited sperm-egg binding in the HZA. These results show that r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) are of strong immunogenicity. They can be used to develop a kit for detecting whether there are autoantibodies to zona pellucida in unexplained infertile women, and their antisera might be useful tools for determining minimal B-cell epitope sequences of several known huZP3 epitope peptides.

Localization and functional importance of a conserved zona pellucida 2 protein domain in the human and bovine ovary using monoclonal anti-ZP2 peptide antibodies.

In mammals, gamete recognition and sperm binding to the oocyte are mediated by the zona pellucida (ZP), an acellular coat surrounding the plasma membrane of the oocyte that consists of particular ZP proteins. The ZP2 protein mediates secondary sperm binding to the ZP. Its primary structures are highly conserved as revealed by cDNA cloning. In the present study, we investigated the localization of ZP2 in human and bovine ovaries and oocytes and the influence of monoclonal anti-ZP2 peptide antibodies upon bovine sperm-egg interactions. We generated a monoclonal anti-ZP2 synthetic peptide antibody, mAb ZP2-20, against a sequence that is strongly conserved in the mammalian ZP2 amino acid sequence. Specificity of mAb ZP2-20 was determined by ELISA and immunoblotting, respectively. Our results show that mAb ZP2-20 specifically detected the peptide used as an antigen and reacted with its corresponding protein antigen in human and bovine ovaries. In order to elucidate effects of mAb ZP2-20 upon bovine sperm-ZP binding, we used the competitive hemizona assay (cHZA) and found that the antibodies clearly inhibit sperm binding to the ZP. We conclude that (i). monoclonal antibodies against ZP2 peptides react with ZP proteins present in bovine and human ovaries and can be used as a specific marker for ZP2; and that (ii). mAb ZP2-20 detects a ZP2 epitope that is of functional relevance for sperm-ZP interactions.

In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen.

Semen diluents containing egg yolk as a cryoprotectant may pose hygienic risks and are difficult to standardize. Although a new generation of semen diluents free of animal ingredients is available, egg yolk-containing extenders are still widely used for cryopreserving semen. We compared the effects of using different extenders on bovine sperm function in vitro and on fertility in vivo. A soy lecithin extender (SL; AndroMed) and an egg yolk-containing (TRIS-EY) extender were tested. No differences (P>0.05) were detected between the two extenders for sperm-zona pellucida binding capacity (HZI=115+/-13). Assessment of the inducibility of the acrosome reaction with progesterone showed no differences (P>0.05) between extenders for live acrosome-reacted sperm (15+/-2.36 and 14.42+/-2.02%, respectively, for SL and TRIS-EY). However, post-thaw sperm motility was significantly lower (P<0.05) when semen was extended in the TRIS-EY diluent. Field trials revealed that nonreturn rates of SL-extended semen showed significantly higher insemination success (P<0.0001) compared with the nonreturn rates for the TRIS-EY extender (70.45 and 67.85%, respectively). We suggest that consistent with quality standards that should be required for cryoprotectant media and because of the superior quality of the egg yolk-free extender, a defined soybean lecithin-containing diluter might be the better choice as a semen extender in the future.

Expression of the Bcl-2 family genes and complexes involved in the mitochondrial transport in prostate cancer cells.

Alteration of molecular pathways triggering apoptosis gives raise to various pathological tissue processes, such as tumorigenesis. The mitochondrial pathway is regulated by both the genes of the Bcl-2 family and the genes encoding mitochondrial transport molecules. Those proteins allow a release of cyctochrome c through the outer mitochondrial membrane. This release activates the caspase cascade resulting in death of cells. There are at least two main transport systems associated with the family of Bcl-2 proteins that are involved in transport of molecules through the outer mitochondrial membrane, i.e., the voltage dependent anion channels (VDACs) and translocases of the outer mitochondrial membrane proteins (TOMs). We investigated the expression of genes of the Bcl-2 family, i.e., pro-apoptotic Bak and Bid, and anti-apoptotic Bcl-2; VDAC gene, i.e., VDAC1, VDAC2 and VDAC3; and TOMM genes, i.e., TOMM20, TOMM22 and TOMM40. This study was performed at the mRNA and the protein level. Fourteen paraffin embedded prostate cancer tissues and five normal prostate tissues were analyzed by the quantitative PCR array and immunohistochemistry. We found a significant increase in both mRNA expression of the anti-apoptotic Bcl-2 gene and VDAC1 gene in prostate cancer tissue in comparison with their normal counterparts. Translation of the anti-apoptotic Bcl-2 and VDAC1 genes in prostate cancer tissue was slightly increased. We observed no significant differences in the mRNA expression of the pro-apoptotic Bak and Bid genes, VDAC2 or VDAC3 genes or the three TOMM genes in these tissues. The pro-apoptotic Bax protein was downtranslated significantly in secretory cells of prostate cancer as compared to normal prostate. We suggest that this protein is a good candidate as biomarker for prostate cancer.

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

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.

Molecular and functional characterization of VDAC2 purified from mammal spermatozoa.

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.

Localisation and function of voltage-dependent anion channels (VDAC) in bovine spermatozoa.

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.

Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum.

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.

Essential role of ZP molecules in tubal transport of embryos in mice.

Our understandings of the molecular and cellular mechanisms underlying tubal transport of embryos are poor. This study describes the essential role of the molecules on the zona pellucida (ZP) in the tubal transport of mouse embryos. The bovine and porcine embryos that were interspecifically transferred to the mouse oviduct were selectively retained in the oviduct and rarely transported to the uterus. Antiserum ZP3-9 against synthetic peptides that are specific for mouse ZP3, significantly interfered with tubal transport of the treated embryos. The treatment of mouse embryos with antiserum ZP2-20 against the synthetic peptides, deduced from the sequences that are conserved in the structure of ZP2 from mouse and human, also inhibited their tubal transport. Among various proteolytic and glycosidic enzymes, treatments with trypsin and beta-glucosidase prior to transfer to the oviduct, significantly interfered with the tubal transport of the enzyme-treated mouse embryos. We hypothesize that species-specific epitopes on the ZP may be recognized by the oviductal cilia and/or the epithelial cells of ducts for tubal transport.