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The influence of estrogens on mouse sperm capacitation and acrosome reaction in vitro.
Tejnická, Magda ; Komrsková, Kateřina (advisor) ; Maňásková, Pavla (referee)
- 7 - Abstract There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by...
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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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Detection of mannosidase in the porcine urogenital tract – study of the sperm releasing from oviductal reservoir
Maňásková, Pavla ; Ren, Š. ; Jelínková, Jitka ; Krejčová, T. ; Liberda, J.
One of the most important steps of reproduction process is the meeting of sperm with oocyte. Binding of sperm with oviductal cells maintains spermatozoa in fertile state. The beginning of sperm capacitation is associated with oocyte ovulation resulting in the sperm release from oviductal reservoir. Hormonal changes after ovulation probably induce distinct oviductal secretion leading to disruption of the sperm protein binding with oviductal saccharide moieties. Another eventuality of the sperm releasing from oviductal reservoir is a change of oviductal environment caused by components of follicular fluid transported with oocyte after ovulation. In the pig, previous studies indicate lectin-type interaction of sperm protein receptors by mannose structures on the surface of oviductal cells. Our study was focused on enzymatic activity of mannosidase and its detection in porcine oviduct (fluid and tissue) and follicular fluid during hormonal cycle. In fluid from follicles in early and late hormonal stages, we measured mannosidase activity by colorimetric methods at physiological and acidic pH. Expression of secreted mannosidase was studied by specific antibody in follicular and oviductal fluids, and oviductal tissues during hormonal cycle. Clearly increased enzymatic activity of secreted mannosidase was found as specific-species in porcine fluid from follicles in late stage of hormonal cycle. On the other hand, detection of secreted mannosidase in follicular fluid as well as in oviductal fluid did not shown any significant differences during hormonal cycle. In oviductal isthmic tissue, we detected decreased protein expression of secreted mannosidase at middle and late follicular phases. These results suggest possible role of follicular mannosidase rather than oviductal one in the sperm releasing from oviductal reservoir in the pig. The additional study of the gene expression of secreted form of mannosidase in oviductal tissue during hormonal cycle should be necessary.
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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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