Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm

Reproductive Biology and Endocrinology

Research

Anatoly Philimonenko¹  Pavel Hozak¹  Hasmik Margaryan²  Andriy Dorosh²  Jana Capkova²  Pavla Manaskova-Postlerova²  Jana Peknicova² 

[1] Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic;Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v. v. i, Videnska 1083, 142 20, Prague 4, Czech Republic;

关键词: Monoclonal antibodies;    Spermatozoa;    GAPDHS;    Immunolabelling;    Transmission electron microscopy;    in vitro;   

DOI  :  10.1186/s12958-015-0008-1

 received in 2014-07-09, accepted in 2015-02-12,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundSperm 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.MethodsIndirect 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.ResultsMonoclonal 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.ConclusionGAPDHS 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.

【 授权许可】

Unknown   
© Margaryan et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.