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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P253 | DOI: 10.1530/repabs.1.P253

Panel of MABs to sperm surface proteins as tool for monitoring of sperm-zona pellucida receptors localization and identification

Michal Zigo1,2, Andriy Dorosh1, Alžběta Póhlová1,2, Věra Jonáková1, Miroslav Sulc2,3 & Pavla Postlerová1

1Laboratory of Reproductive Biology, Institute of Biotechnology, The Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic; 2Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague 2, Czech Republic; 3Institute of Microbiology, The Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic.

Introduction: Primary binding of the sperm to the zona pellucida (ZP), or its analogues, is one of the many steps necessary for successful fertilization in all sexually reproducing species. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Primary-binding receptors are localized throughout the acrosomal region of the sperm surface of which many have been disclosed in various mammals.

Materials and methods: Panel of MABs against proteins from the sperm surface was prepared. Antibodies were screened by imunoflourescence and western blotting for protein localizations and competence of antibodies respectively. Proteins localized on the sperm head and simultaneously detected by western blot were further studied in terms of imunolocalization in reproductive tissues and fluids, binding to ZP, imunoprecipitation and sequencing.

Results and discussion: Out of 17 prepared antibodies, eight antibodies were recognizing proteins localized on the sperm head and also detecting proteins of interest by western blotting. Further only three antibodies recognized proteins which also coincided in binding to ZP. These three antibodies were used for imunoprecipitation, and further protein sequencing of imunoprecipitates revealed that the antibodies distinguish acrosin precursor, RAB2A protein, and lactadherin P47. Acrosin and lactadherin P47 have been already detected on the sperm surface and their physiological functions in reproduction have been proposed. To our knowledge, this is the first time RAB2A has been found on the surface of sperm. This work was supported by GACR P503/12/1834, P502/14/05547S, UNCE204025/2012, RVO61388971, and by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

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