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Reproductive Biology and Endocrinology
Published in: Reproductive Biology and Endocrinology 1/2015

Open Access 01-12-2015 | Research

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

Authors: Hasmik Margaryan, Andriy Dorosh, Jana Capkova, Pavla Manaskova-Postlerova, Anatoly Philimonenko, Pavel Hozak, Jana Peknicova

Published in: Reproductive Biology and Endocrinology | Issue 1/2015

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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.

Conclusion

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.
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Metadata
Title
Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm
Authors
Hasmik Margaryan
Andriy Dorosh
Jana Capkova
Pavla Manaskova-Postlerova
Anatoly Philimonenko
Pavel Hozak
Jana Peknicova
Publication date
01-12-2015
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2015
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-015-0008-1

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