title: Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.
abstract: BACKGROUND
  Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay.
  
  
  METHODS
  Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay.
  
  
  RESULTS
  Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding.
  
  
  CONCLUSIONS
  GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.
authors.affiliation: Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. hasmik.margaryan@ibt.cas.cz.
authors.forename: Hasmik
authors.initials: H
authors.lastname: Margaryan
authors.affiliation: Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. andriy.dorosh@ibt.cas.cz.
authors.forename: Andriy
authors.initials: A
authors.lastname: Dorosh
authors.affiliation: Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. jana.capkova@ibt.cas.cz.
authors.forename: Jana
authors.initials: J
authors.lastname: Capkova
authors.affiliation: Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. pavla.postlerova@ibt.cas.cz.
authors.forename: Pavla
authors.initials: P
authors.lastname: Manaskova-Postlerova
authors.affiliation: Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. anatoly.philimonenko@img.cas.cz.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Philimonenko
authors.affiliation: Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. pavel.hozak@img.cas.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Hozak
authors.affiliation: Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic. jana.peknicova@ibt.cas.cz.
authors.forename: Jana
authors.initials: J
authors.lastname: Peknicova
mesh_terms: D000177:Acrosome / Q000378:metabolism; D000818:Animals; D004734:Energy Metabolism; D005407:Flagella / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D013081:Sperm Motility; D013084:Sperm-Ovum Interactions; D013091:Spermatogenesis; D013094:Spermatozoa / Q000378:metabolism*; D013552:Swine / Q000378:metabolism; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: D005987:Glyceraldehyde-3-Phosphate Dehydrogenases