AIMTo investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa.

METHODSThe 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.

RESULTSImmunodetection 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.

CONCLUSIONOur 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.

Acrosome Reaction ; drug effects ; physiology ; Animals ; Cattle ; Enzyme Inhibitors ; pharmacology ; Genistein ; pharmacology ; Male ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Sperm Capacitation ; drug effects ; physiology ; Zona Pellucida ; drug effects ; physiology

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.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Immune Sera ; immunology ; Immunization ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sperm-Ovum Interactions ; immunology ; Zona Pellucida Glycoproteins