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Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm
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SYSNO ASEP 0443992 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm Author(s) Margaryan, Hasmik (BTO-N) RID
Dorosh, Andriy (BTO-N) RID
Čapková, Jana (BTO-N)
Maňásková-Postlerová, Pavla (BTO-N) ORCID
Philimonenko, Anatoly (UMG-J)
Hozák, Pavel (UMG-J) RID, ORCID
Pěknicová, Jana (BTO-N) RIDNumber of authors 7 Source Title Reproductive Biology and Endocrinology. - : BioMed Central
Roč. 13, č. 15 (2015)Number of pages 9 s. Language eng - English Country US - United States Keywords monoclonal antibodies ; spermatozoa ; GAPDHS ; immunolabeling ; transmission electron microscopy ; in vitro sperm/zona pellucida binding assay Subject RIV CE - Biochemistry OECD category Reproductive biology (medical aspects to be 3) Subject RIV - cooperation Institute of Molecular Genetics - Genetics ; Molecular Biology R&D Projects ED1.1.00/02.0109 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) GAP503/12/1834 GA ČR - Czech Science Foundation (CSF) Method of publishing Open access Institutional support BTO-N - RVO:86652036 ; UMG-J - RVO:68378050 UT WOS 000351428100001 DOI 10.1186/s12958-015-0008-1 Annotation 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. 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. 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. 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 MoAb 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. Workplace Institute of Biotechnology Contact Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Year of Publishing 2016 Electronic address https://rbej.biomedcentral.com/articles/10.1186/s12958-015-0008-1
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