|
|
Study of sperm proteins in different mammalian species
Pohlová, Alžběta ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee)
Reproduction is an essential feature of all animals and a fundamental step to produce new generations. Study of sperm proteins is crucial for understanding of the sperm-egg recognition. We searched out sperm surface proteins involving in the zona pellucida (ZP) binding and studied whether these proteins are preserved throughout mammalian species. Indirect immunofluorescent technique was used to test a panel of monoclonal antibodies prepared against boar sperm surface proteins on spermatozoa of bull and mice. We found a cross-reactivity of some antibodies against boar sperm with bull ejaculated and mouse epididymal spermatozoa. Further, we isolated sperm proteins from different mammalian species, such as pig, bull, dog, cat, mouse and human. Proteins were separated by SDS- electrophoresis and protein/glycoprotein profiles from epididymal, ejaculated and in vitro capacitated sperm were compared. The interaction of sperm with ZP was studied on electrophoretically-separated sperm surface proteins from pig and bull with biotin-labeled ZP glycoproteins. Antibodies, which reacted with boar sperm surface proteins with ZP- binding activity, therefore could be potential egg-binding receptors, were used for monitoring of the sperm protein origin in reproductive fluids and tissues. (In Czech) Keywords: sperm...
|
|
|
Study of sperm proteins in different mammalian species
Pohlová, Alžběta ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee)
Reproduction is an essential feature of all animals and a fundamental step to produce new generations. Study of sperm proteins is crucial for understanding of the sperm-egg recognition. We searched out sperm surface proteins involving in the zona pellucida (ZP) binding and studied whether these proteins are preserved throughout mammalian species. Indirect immunofluorescent technique was used to test a panel of monoclonal antibodies prepared against boar sperm surface proteins on spermatozoa of bull and mice. We found a cross-reactivity of some antibodies against boar sperm with bull ejaculated and mouse epididymal spermatozoa. Further, we isolated sperm proteins from different mammalian species, such as pig, bull, dog, cat, mouse and human. Proteins were separated by SDS- electrophoresis and protein/glycoprotein profiles from epididymal, ejaculated and in vitro capacitated sperm were compared. The interaction of sperm with ZP was studied on electrophoretically-separated sperm surface proteins from pig and bull with biotin-labeled ZP glycoproteins. Antibodies, which reacted with boar sperm surface proteins with ZP- binding activity, therefore could be potential egg-binding receptors, were used for monitoring of the sperm protein origin in reproductive fluids and tissues. (In Czech) Keywords: sperm...
|
|
|
Isolation and characterisation of antimicrobial peptides from reproductive tract of sow and cow
Pohlová, Alžběta ; Liberda, Jiří (advisor) ; Ryšlavá, Helena (referee)
Oviductal, follicular and uterine fluid were isolated from reproductive tract of sow and cow. Obtained fluids were lyophilized and tested for potential antimicrobial activity. Determination of antimicrobial aktivity was tested using cultures of Escherichia coli (serotype K-12). Antimicrobial activity was detected in sow oviductal, follicular and uterine fluid and in bovine oviductal and follicular fluid. Furthermore, an inhibition of E. coli (serotyp K-12) cell growth was also tested, depending on the proteins of bovine oviductal fluid and the amount of E. coli cells in the culture. (In Czech) Keywords: oviductal fluid, follicular fluid, uterine fluid, antimicrobial activity
|
|
|
Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
|
|
|
Epididymal maturation – a crucial step in the post-testicular sperm development
Postlerová, Pavla ; Pohlová, Alžběta ; Zigo, Michal ; Jonáková, Věra
Mammalian spermatozoa after their development in testis undergo the post-testicular maturation in epididymis where acquire their fertilization ability and competence of movement. The epididymis is tissue with very active fluid-absorbing and fluid-secreting activity. Epididymal fluid contains ions and small molecules, proteins, glycoproteins and enzymes. The surface of spermatozoa is exposed directly to the epididymal fluid, and the sperm plasma membrane is significantly changed. Some testicular proteins are altered, masked, or replaced by new proteins/glycoproteins of epididymal origin. Several proteins produced by epididymis have been described in various mammalian species and shown to be associated with spermatozoa suggesting a role in the sperm maturation and/or sperm-egg binding and fusion. We isolated proteins from fluid, tissue and sperm of boar epididymis, and separated them by chromatographic and electrophoretic methods. We searched for known proteins using panel of antibodies and tested proteins of epididymal fluid for binding abilities. In the epididymis, we found proteins described as proteins of seminal plasma and associated with the sperm surface, such as spermadhesins, beta-microseminoprotein and acrosin inhibitor. These proteins were detected in epididymal sperm, fluid and tissue. We showed that some epididymal proteins may bind the spermatozoa and change the binding sites on the sperm surface. We determined and identified some proteins from boar epididymal fluid with affinity to heparin, hyaluronan and zona pellucida glycoproteins. These phenomena indicate that epididymal fluid proteins bind to the sperm surface during epididymal maturation and might subsequently play role in the sperm capacitation or sperm-zona pellucida binding.
|
|
|
Sperm protein profiles of different mammalian species
Pohlová, Alžběta ; Zigo, Michal ; Jonáková, Věra ; Postlerová, Pavla
Proteins are a substantial equipment of the spermatic cell; therefore, the characterization of sperm proteins is crucial for explanation of molecular mechanisms in the reproduction process. We isolated sperm proteins from different mammalian species - pig, bull, human, mouse, dog and cat. Extracted proteins were separated by SDS-electrophoresis and protein/glycoprotein profiles from epididymal or ejaculated sperm were compared. Additionally, we tested cross-reactivity of antibodies prepared to sperm boar proteins on spermatozoa of other mammalian species using immunofluorescent technique. Our future plan is to compare the protein profiles of sperm during their functional development (epididymal, ejaculated, capacitated) in various mammalian species and identify species-specific sperm proteins with zona pellucida binding activity.
|
|
|
Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
|
|
|
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.
|
guest ::
RSS feed.