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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
Seminal plasma proteins bind to the sperm surface at ejaculation and may modulate sperm properties during reproduction. Porcine spermadhesins (AQN, AWN, PSP) are secreted mainly by the seminal vesicles (SV), but their mRNAs have been found also in the cauda epididymis and prostate. Using specific polyclonal antibodies, PSP-I and PSP-II proteins were immunodetected in tissue extracts from cauda epididymis, prostate, SV and Cowper´s glands on the blots, and in secretory tissues of cauda epididymis, prostate and SV by indirect immunofluorescence. We localized PSP spermadhesins on epididymal and ejaculated spermatozoa. PSP proteins are produced not only by SV and prostate, but also by epididymis. Characterization of seminal plasma proteins expressed in the individual reproductive organs might help to understand their role in the reproduction process.
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Role of sperm surface proteins in reproduction
Jonáková, Věra ; Postlerová, Pavla ; Tichá, M. ; Pěknicová, Jana
Seminal plasma proteins bind to the sperm surface at ejaculation and may modulate sperm properties during reproduction. DQH sperm surface protein shows affinity to oviductal epithelium and zona pellucida (ZP) glycoproteins. The mRNA transcript of DQH protein was found in seminal vesicles (SV). DQH was immunodetected by monoclonal antibodies (MAbs) in SV extract and fluid, on SV tissue sections and on the membrane-associated acrosome part of ejaculated spermatozoa. These results confirmed the ability of DQH protein to bind to the sperm surface at ejaculation and to participate in the formation of the sperm reservoir in the porcine oviduct. Moreover, monoclonal antibodies reduced binding of sperm to oocytes and proved the role of DQH protein in the sperm-ZP primary binding.
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Exprese a lokalizace inhibitoru akrosinu v kančím reprodukčním traktu
Jonáková, Věra ; Davidová, Nina ; Maňásková, Pavla
Proteinase inhibitors present in seminal plasma inactivate prematurely released sperm acrosin and protect spermatozoa and reproductive epithelium of the male and female reproductive tracts against proteolytic degradation. Acrosin inhibitor mRNA was detectable in epididymis, seminal vesicles, prostate and Cowperś glands. By indirect immunofluorescence, acrosin inhibitors were detected in the secretory tissues of cauda epididymis, prostate and SV, and on epididymal and ejaculated spermatozoa in the acrosomal region. The presence of inhibitors of the proteolytic enzymes in epididymis is very important for the regulation of the sperm membrane protein processing.
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Původ proteinů kančí semenné plazmy
Maňásková, Pavla ; Kyselová, Vendula ; Tichá, M. ; Jonáková, Věra
Spermadhesins were detected with their antibodies in boar epididymal fluid, but DQH and beta-microseminoprotein were not. Indirect immunofluorescence technique was employed to determine the distribution of seminal plasma proteins on tissue sections of the reproductive organs. The mRNA of the DQH was found only in seminal vesicles, not in the epididymis, and the mRNA of beta-MSP in testis, seminal vesiclas and prostate.
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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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