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Panel of monoclonal antibodies – alternative tool For monitoring of sperm–zona pellucida receptors localization and identification
Zigo, Michal ; Dorosh, Andriy ; Pohlová, Alžběta ; Jonáková, Věra ; Šulc, Miroslav ; Maňásková-Postlerová, Pavla
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.
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Enzymatic and inhibiting activity in boar epididymal fluid
Davidová, Nina ; Ren, Š. ; Liberda, J. ; Jonáková, Věra ; Maňásková-Postlerová, Pavla
Sperm maturation, represents a key step in the reproduction process. Spermatozoa, particularly the plasma membrane, are exposed to epididymal fluid (EF) components representing the natural environment essential for their post-testicular maturation. Changes in the sperm membrane proteins are influenced by proteolytic and glycosidic enzymes present in the EF. Accordingly, the occurrence of inhibitors in this reproductive organ is very important for the regulation of sperm membrane protein processing. In present study, we monitored protease and glycosidase activities, and inhibitors of metallo- and serine proteinases in boar EF. Additionally, we studied acrosin inhibitor in fluid, spermatozoa and tissue along the epididymis. We chromatographically separated boar EF into several fractions. These fractions were subjected to SDS-electrophoresis and the separated proteins were either studied by zymographic methods or transferred to nitrocellulose membranes for detection of metallo- and serine proteinases and their inhibitors, and acrosin inhibitor by specific antibody, respectively. Acrosin inhibitor was monitored also in the sperm and tissue of the boar epididymis. In boar epididymal fluid, several metallo- and serine proteinases with different molecular masses, and inhibitors of metalloproteinase MMP-9 and acrosin were found. We measured strong activity of mannosidase in this fluid. Using specific antibody, we registered the increasing signal of acrosin inhibitor from caput to cauda epididymis in the spermatozoa, fluid and also tissue. Proteinases and their inhibitors in reproductive fluids may play a significant role in reproduction processes. Especially, acrosin inhibitor in the reproductive tract inactivates prematurely released sperm acrosin and protects spermatozoa and reproductive epithelium against proteolytic degradation. High mannosidase activity in boar EF suggests evident role of mannose structures in the sperm interaction during reproductive events.
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Povrchové proteiny spermie: původ, biochemické vlastnosti a úloha v reprodukci
Jonáková, Věra ; Postlerová, Pavla ; Tichá, M. ; Pěknicová, Jana
Proteiny semenné plasmy se váží na povrch spermie při ejakulaci a modulují vlastnosti spermie při reprodukci. Kančí spermadhesiny (AQN, AWN, PSP) jsou sekretovány hlavně semenými vaky (SV), ale jejich mRNA byla nalezena také v cauda epididymis a prostatě. Pomocí specifických protilátek PSP I a PSP II proteiny byly imunodetekovány na blotu v tkáňových extraktech cauda epididymis, prostatě, SV, a nepřímou imunofluorescencí v sekreční tkáni cauda epididymis, prostatě, SV. Lokalizovali jsme PSP spermadhesiny na epididymálnícj a ejakulovaných spermiích. PSP proteiny jsou produkovány SV, prostatou a také nadvarletem. Charakterizace proteinů semenné plasmy exprimovaných v jednotlivých reprodukčních orgánech napomůže pochopit jejich úlohu v reprodukci
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Role povrchových proteinů v reprodukci
Jonáková, Věra ; Postlerová, Pavla ; Tichá, M. ; Pěknicová, Jana
Během ejakulace se proteiny semenné plasmy navazují na povrch spermie a tvoří povrchové proteiny spermie, které mohou změnit vazebné vlastnosti spermie. DQH povrchový protein spermie vykazuje afinitu k oviduktálnímu epitelu a glykoproteinům zona pellucida (ZP). DQH mRNA byla detekována v semenných vacích (SV). DQH byl imunodetekován monoklonálními protilátkami (Mabs)v extraktu a tekutině i na tkáňových řezech SV vaků a na plasmatické membráně akrosomu ejakulovaných spermií. Výsledky potvrzují schopnost DQH proteinu se vázat na povrch spermie při ejakulaci, účast při tvorbě rezervoáru spermií v prasečím oviduktu. Spermie preinkubované s Mabs proti DQH proteinu před koinkubací s vajíčkem redukují vazbu spermie k vajíčku. Pro primární vazbu spermie s ZP glykoproteiny vajíčka je důležitý intaktní DQH protein na povrchu spermie.
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Exprese a lokalizace inhibitoru akrosinu v kančím reprodukčním traktu
Jonáková, Věra ; Davidová, Nina ; Maňásková, Pavla
Inhibitory proteinas v semenné plasmě inhibují předčasně uvolněnou spermiovou proteinasu akrosin a ochraňují tak spermie a epitel samčího a samičího traktu proti proteolyse. Akrosin inhibitor mRNA byla nalezena v epididymu, v semených vacích, prostatě a Cowperových žlazách. Nepřímou imunofluorescencí inhibitor akrosinu byl detekován v sekreční tkáni cauda epididymu, prostatě a semených vacích a v akrosomální oblasti na epididymálních a ejakulovaných spermií. Přítomnost inhibitorů proteolytických enzymů v epididymu je důležitá pro processing spermiových membránových proteinů.
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Characterization of peptide proteinase inhibitors isolated from boar seminal plasma
Jelínková, Petra ; Tichá, M. ; Jonáková, Věra
Most of proteins of boar seminal plasma are present in this medium under physiological conditions in the form of aggregates and not of monomers. In the present communication we have shown that not only spermadhesins, but also proteinase inhibitors participate in the formation of associated complexes. Proteinase inhibitors were found after gel chromatographic separation mainly in the fraction with the lowest relative molecular mass. Two different types of serine proteinase inhibitors were isolated from boar seminal plasma ů (i) glycosylated inhibitor with Mr 12 kDa, (ii) non-glycosylated sperm-associated acrosin inhibitor with Mr 8 kDa, and a new type of low molecular weight protein that is probably composed of two subunits coupled by the -S-S- bridge. The proteinase inhibitor with Mr 8 kDa was proved to form a heterodimer with AQN 1 spermadhesin under physiological conditions, whereas inhibitor with Mr 12 kDa forms homodimers.
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