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Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm–zona pellucida receptors

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Abstract

Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-defined sequential process. Primary binding receptors, many of which have been disclosed in various mammals, are localized throughout the acrosomal region of the sperm surface. A panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence for protein localization and Western blotting. Proteins localized on the sperm head and simultaneously detected by Western blotting were further studied in terms of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and sequencing. Of 17 prepared antibodies, 8 recognized proteins localized on the sperm head and also detected proteins of interest by Western blotting. Only three other antibodies recognized proteins that also coincided in binding to ZP. These three antibodies were used for immunoprecipitation, and further protein sequencing of immunoprecipitates revealed that these antibodies distinguished acrosin precursor, RAB-2A protein, and lactadherin P47. This is not the first time we have detected acrosin on the surface of ejaculated and capacitated sperm. However, to our knowledge, this is the first time RAB-2A has been detected on the sperm surface. Lactadherin P47 has already been characterized and its physiological function in reproduction has been proposed.

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Acknowledgments

This work was supported by grants Nos. P503/12/1834 and P502/14/05547S of the Grant Agency of the Czech Republic, by Charles University in Prague (UNCE204025/2012), by the Institutional Research Project RVO61388971, and by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

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The authors declare no conflicts of interest.

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  1. Laboratory of Reproductive Biology, Institute of Biotechnology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic

    Michal Zigo, Andriy Dorosh, Alžběta Pohlová, Věra Jonáková & Pavla Maňásková-Postlerová

  2. Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague 2, Czech Republic

    Michal Zigo, Alžběta Pohlová & Miroslav Šulc

  3. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic

    Miroslav Šulc

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  1. Michal Zigo
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Correspondence to Michal Zigo.

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Zigo, M., Dorosh, A., Pohlová, A. et al. Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm–zona pellucida receptors. Cell Tissue Res 359, 895–908 (2015). https://doi.org/10.1007/s00441-014-2072-9

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