Panel of monoclonal antibodies – alternative tool For monitoring of sperm–zona pellucida receptors localization and identification

0428725 - BTÚ 2015 CZ eng K - Konferenční příspěvek (tuzemská konf.)
Zigo, Michal - Dorosh, Andriy - Pohlová, Alžběta - Jonáková, Věra - Šulc, Miroslav - Maňásková-Postlerová, Pavla
Panel of monoclonal antibodies – alternative tool For monitoring of sperm–zona pellucida receptors localization and identification.
Book of abstracts XXth Symposium of Biology and Immunology of Reproduction with international participation. Praha: Biotechnologický ústav AVČR, v.v.i, 2014, s. 1-82
[XXth Symposium of Biology and Immunology of Reproduction with international participation. Třešť (CZ), 22.05.2014-24.05.2014]
Grant CEP: GA ČR(CZ) P503/12/1834; GA ČR(CZ) P502/14/05547S; GA MŠMT(CZ) CZ1.05/1.1.00/02.0109
Výzkumný záměr: CEZ:AV0Z50520701
Institucionální podpora: RVO:86652036 ; RVO:61388971
Klíčová slova: monoclonal antibody * zona pellucida * acrosin precursor * RAB2A * lactahedrin P47
Kód oboru RIV: CE - Biochemie

Primary binding of the sperm to the zona pellucida (ZP) 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. For the monitoring sperm-zona pellucida receptors in terms of localization and characterization - panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence and Western blotting for protein localizations and competence of antibodies, respectively. Antibodies recognizing proteins localized on the sperm head and simultaneously detected by Western blot were further studied by means of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and protein identification using MS analysis. Out of 17 prepared antibodies, 8 antibodies were simultaneously recognizing proteins localized on the sperm head and detecting proteins of interest by Western blotting. Further only 3 antibodies recognized proteins which also coincided in binding to ZP. These 3 antibodies were used for immunoprecipitation, and further protein identification of immunoprecipitates 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 and its physiological function in the process of fertilization remains undisclosed.
Trvalý link: http://hdl.handle.net/11104/0235849