Number of found documents: 46
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Book of abstract of XXIInd Symposium of immunology and biology of reproduction
Pěknicová, Jana; Elzeinová, Fatima; Kubátová, Alena
2016 - English
Book of abstracts. The Symposium was focused on immunology of reproduction and specific problems in reproduction (mainly in human infertility). Keywords: spermatology; biochemistry; cell interaction; monoclonal antibody; sterility/fertility; gene expression Available at various institutes of the ASCR
Book of abstract of XXIInd Symposium of immunology and biology of reproduction

Book of abstracts. The Symposium was focused on immunology of reproduction and specific problems in reproduction (mainly in human infertility).

Pěknicová, Jana; Elzeinová, Fatima; Kubátová, Alena
Biotechnologický ústav, 2016

Recombinant proteins and their usage in the studies of male gamete function
Dorosh, Andriy; Žatecká, Eva; Pěknicová, Jana; Dvořáková-Hortová, Kateřina
2016 - English
Keywords: recombinant protein; spermatogenesis; glycolytic enzyme Available at various institutes of the ASCR
Recombinant proteins and their usage in the studies of male gamete function

Dorosh, Andriy; Žatecká, Eva; Pěknicová, Jana; Dvořáková-Hortová, Kateřina
Biotechnologický ústav, 2016

Biochemické motody jako nástroj pro studium proteinů v reprodukci
Postlerová, Pavla; Zigo, Michal; Pohlová, Alžběta; Jonáková, Věra
2016 - Czech
Studium molekulárních mechanismů rerodukce je důležité pro pochopení tohoto děje. pro extrakci proteinů ze spermií využíváme různé přístupy a specializované kity, které izolují proteiny z různých částí a povrchu spermatické buňky. Proteiny z reprodukčních tekutin dělíme pomocí chromatografických metod. Charakterizaci a porovnání proteinů získaných odlišnými extrakčními metodami nebo proteinů různých savčích druhů a proteinů spermií, které jsou v rozdílných stádiích svého vývoje, provádíme pomocí SDS a 2D elektroforézy. Elektroforeticky separované proteiny přenesené na NC membránu využíváme pro detekci pomocí protilátek nebo k vazebnýcm studiím s biotinem značenými ligandy. Izolace proteinů z reprodukčních tkání a tekutin a jejich detekce pomocí protilátek je nezbytná pro určení původu proteinů reprodukčního traktu. Lokalizace proteinů na povrchu a uvnitř spermatických buněk a v sekreční tkáni reprodukčních orgánů studujeme pomocé imunofluorescenční mikroskopie. Cílená izolace určitých proteinů spermatické buňky, jejich lokalizace na spermiích nebo v tekutinách a tkáních reprodukčního traktu slouží jako nepostradatelné nástroje pro studium molekulárních mechanismů reprodukčního procesu. 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. Keywords: protein extraction; chromatography; ligands; sperm surface proteins Available at various institutes of the ASCR
Biochemické motody jako nástroj pro studium proteinů v reprodukci

Studium molekulárních mechanismů rerodukce je důležité pro pochopení tohoto děje. pro extrakci proteinů ze spermií využíváme různé přístupy a specializované kity, které izolují proteiny z různých ...

Postlerová, Pavla; Zigo, Michal; Pohlová, Alžběta; Jonáková, Věra
Biotechnologický ústav, 2016

Biotechnologie ve světě a českých zemích
Pěknicová, Jana
2016 - Czech
Biotechnologie je jakákoli technologie využívající biologické systémy, živé organismy nebo jejich části k určité výrobě nebo jejich přeměně či jinému specifickému použití. Přednáška by měla ukázat stručný přehled o biotechnologiích ve světě, v českých zemích, v Biotechnologickém ústavu AVČR, v.v.i. a v projektu BIOCEV. 1. Biotechnologie ve světě: počátek biotechnologií - mezníky- aplikace v lékařství - genová terapie - geneticé modifikace (věda , zemědělství, průmysl) 2. biotechnologie v českých zemích: česká biotechnologie: mezníky 3. Biotechnologický ústav AVČR, v.v.i.: výzkum a výstupy do praxe 4. BIOCEV: biotechnologické a biomedicínské centrum Akademie věd a Univerzity Karlovy ve Vestci. - Ustavení centra excelence jako součást evropské vědecké oblasti zaručující vývoj moderních biotechnologií a medicíny. Biotechnology in the world and the Czech lands Biotechnology is any technology that uses biological systems, living organisms, or parts of a particular production or their conversion or other specific applications. The lecture should show a brief overview of the biotechnology industry in the world, in the Czech lands, in the Institute of Biotechnology CAS, v. v. i., and in the project BIOCEV: 1. Biotechnology in the world • Beginning of biotechnologies • Milestones • Application in medicine • Gene therapy • Genetic modification (science, agriculture, industry) 2. Biotechnology in the Czech lands • Czech biotechnology: milestones 3. Institute of Biotechnology CAS, v. v. i. • Research and outputs into practice 4. BIOCEV – Biotechnological and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec Establishment of centre of excellence as part of the European scientific area guaranteeing the development of modern biotechnology and medicine. Keywords: biotechnology; gene therapy; genetic modification Available at various institutes of the ASCR
Biotechnologie ve světě a českých zemích

Biotechnologie je jakákoli technologie využívající biologické systémy, živé organismy nebo jejich části k určité výrobě nebo jejich přeměně či jinému specifickému použití. Přednáška by měla ukázat ...

Pěknicová, Jana
Biotechnologický ústav, 2016

Epididymal maturation – a crucial step in the post-testicular sperm development
Postlerová, Pavla; Pohlová, Alžběta; Zigo, Michal; Jonáková, Věra
2016 - English
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. Keywords: sperm maturation; glycoproteins; spermadhesins; epididymal fluid; monoclonal antibodies Available at various institutes of the ASCR
Epididymal maturation – a crucial step in the post-testicular sperm development

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 ...

Postlerová, Pavla; Pohlová, Alžběta; Zigo, Michal; Jonáková, Věra
Biotechnologický ústav, 2016

Estrogen receptor beta (ERβ) in testicular cells and sperm
Dostálová, Pavla; Žatecká, Eva; Děd, Lukáš; Dorosh, Andriy; Postlerová, Pavla; Jonáková, Věra; Dvořáková-Hortová, Kateřina; Pěknicová, Jana
2016 - English
Estrogen is a steroid hormone that plays an important role during sperm development in the male and female reproductive tract. Estrogen signalling is a complex process that depends on cell milieu and presence of receptors. Thanks to the steroid nature of estrogens, they can pass through the plasmatic membrane and bind to the intracellular estrogen receptors (ERs). Within the cell, there are several pools of ERs. One of them is localized to the cell nucleus and their activation leads to direct or indirect binding to DNA and ultimately to alternation in gene expression (genomic pathway). Other pools of ERs are associated with plasma membrane or are located in cytosol. Activation of membrane associated ERs leads to rapid non-genomic responses. Nowadays, two classical estrogen receptors are known – ERα and ERβ. Since ERβ is a predominant variant in testes, we focused our study on expression of ERβ variants in murine testes and sperm. We detected two variants of ERβ at mRNA level in both, testes and sperm. These variants differ in 54 nucleotids within the ligand binding domain and this variability results in different affinity to estrogen. We analyzed individual testicular cell types (spermatogonia, spermatocytes, spermatids, Sertoli cells) by RT-qPCR. Our results suggest that both ERβ variants are coexpressed in the same cell type and may therefore interact together. This may have consequences in mediating of estrogen signalling. Moreover, ERβ is expressed more in the later stages of spermatogenesis suggesting the role of ERβ in these stages or alternatively in spermatozoa alone. At the protein level, we detected ERβ in nuclear, membrane and cytosolic fraction prepared from testicular tissue suggesting the involvement of both, genomic and non-genomic, pathways of estrogen signaling in testes. In sperm, anti-ERβ antibodies localized ERβ in acrosome region and tail which is in accordance with the known role of estrogen on capacitation, acrosome reaction and motility. Keywords: estrogen receptor; gene expression; testicular cell types; RT-qPCR; monoclonal antibodies Available at various institutes of the ASCR
Estrogen receptor beta (ERβ) in testicular cells and sperm

Estrogen is a steroid hormone that plays an important role during sperm development in the male and female reproductive tract. Estrogen signalling is a complex process that depends on cell milieu and ...

Dostálová, Pavla; Žatecká, Eva; Děd, Lukáš; Dorosh, Andriy; Postlerová, Pavla; Jonáková, Věra; Dvořáková-Hortová, Kateřina; Pěknicová, Jana
Biotechnologický ústav, 2016

Exprese vybraných proteinů spermií u mužů s normálním a patologickým spermiogramem za použití monoklonálních protilátek
Pěknicová, Jana; Čapková, Jana; Dorosh, Andriy; Margaryan, Hasmik; Kubátová, Alena; Děd, Lukáš
2016 - Czech
Nedávné studie ukázaly že neplodnost v lidské populaci postihuje odhadem 15% párů v reprodukčním věku. Mužská neplodnost je primární příčinou u 60% těchto případů. Z těchto důvodů jsme analyzovali povrchové a akrozomální proteiny spermií u mužů s normálním a patologickým spermiogramem. Zjistili jsme že intra-akrozomální proteiny: TERA, GAPDHS, PRKAR2A, které lze analyzovat pomocí našich monoklonálních protilátek , jsou rozdílně exprimovány u zdravých mužů a mužů s asthenozoospermií a to se signifikantně sníženou expresí u asthenozoospermie. tyto proteiny se účastní energetického metabolismu a apoptózy buněk a některé z nich i vazby na vajíčko - mají tedy důležitou roli v reprodukci. Na druhou starnu nebyly zjisštěny statisticky významné rozdíly v expresi povrchových proteinů (Appolipoprotein J, Semenogelin). Naše závěry ukazují že asthenozoospermie jako komplexní poruch apermatu je často v kombinaci s jinými patologickými stavy, které nejsou diagnostikovány základní analýzou spermatu. Monoklonální protilátky jsou tak vhodným nástrojem pro detekci proteinů spojených s patologií spermií ve spermatu s nízkou pohyblivostí spermií. Obecně, monoklonální protilátky proti proteinům spermie jsou vhodným nástrojem k detekci kvality spermií v reprodukční medicíně. Recent studies show that infertility in human populations it affects an estimated 15% of couples of reproductive age. Male infertility is the primary cause for 60% of these cases. For these reasons, we analyzed the acrosomal and sperm surface proteins in men with normal and pathological spermiograms. We found that intra-acrosome proteins: TERA (Transitional endoplasmic reticulum ATPase), GAPDHS (Sperm Glyceraldehyde-3-phosphate dehydrogenase), and PRKAR2A (C-AMP-dependent protein kinase II, PRKAR2A), which can be identified using our monoclonal antibodies, are different express in healthy men and men with astenozoospermia (with reduced sperm motility), and with a significantly reduced expression in the astenozoospermia. These proteins are involved in energy metabolism and apoptosis of the cells, and some of them in the sperm-egg interaction; therefore, they have an important role in reproduction. On the other hand, there were no statistically significant differences in the expression of surface proteins (Appolipoprotein J (Clusterin) and Semenogelin). Our findings show that astenozoospermia as a complex disorder of the semen is often combined with other pathological conditions that are not diagnosed by the semen analysis. Therefore monoclonal antibodies are so suitable instrument for the detection of proteins associated with the pathology of the sperm in the semen with low sperm motility. In general, monoclonal antibodies against the sperm proteins are an appropriate tool to detect sperm quality in reproductive medicine. Keywords: sperm acrosomal proteis; sperm surface proteins; monoclonal antibody; asthenozoospermia; normozoospermia Available at various institutes of the ASCR
Exprese vybraných proteinů spermií u mužů s normálním a patologickým spermiogramem za použití monoklonálních protilátek

Nedávné studie ukázaly že neplodnost v lidské populaci postihuje odhadem 15% párů v reprodukčním věku. Mužská neplodnost je primární příčinou u 60% těchto případů. Z těchto důvodů jsme analyzovali ...

Pěknicová, Jana; Čapková, Jana; Dorosh, Andriy; Margaryan, Hasmik; Kubátová, Alena; Děd, Lukáš
Biotechnologický ústav, 2016

Single cell expression analysis of genes with potential mrna gradient in mouse oocytes
Dorosh, Andriy; Margaryan, Hasmik; Vodička, Martin; Ergang, Peter; Šídová, Monika; Dvořáková-Hortová, Kateřina
2016 - English
In frogs, there are clearly visible differently pigmented animal and vegetal poles of the egg determined before fertilization and leading to asymmetrical divisions. Mammalian egg does not show any comparable differentiation and it has been generally accepted that even the individual blastomeres in 2-cell and 4-cell embryos are homogenous. However, recent findings suggest that those blastomeres display different gene expression patterns and might already possess some inclinations to specific cell lineages. We therefore raised a question, whether there could be any mRNA or protein gradients in pre-fertilization oocytes similar to a previously described amphibian egg one. In mammalian eggs, there is a membrane region that is poor in microvilli, cortical granules are absent beneath plasma membrane and sperm cells generally do not bind to this location. This microvilli free region also covers the egg nucleus, and cytoskeleton localization differs markedly to the rest of the cortical space, forming actin –myosin II cortical cap/ring and is considered as animal pole. The purpose of this study was to determine gene products that can be detected at single cell level using qPCR and display gradient like distribution in mature oocytes. We checked expression of 12 selected genes in a pool of 10 oocytes and single mature oocytes. Then, we analysed gene expression in fixed intact oocytes and those undergoing laser capture microdissection procedure (LCMD). Eventually, we have determined six candidate genes for the study of intracellular spatial gene expression in mature mammalian oocytes by subcellular qPCR and in situ hybridization. Keywords: single cell expresion analysis; RT-qPCR; oocytes; Laser capture microdissection; in situ hybridization Available at various institutes of the ASCR
Single cell expression analysis of genes with potential mrna gradient in mouse oocytes

In frogs, there are clearly visible differently pigmented animal and vegetal poles of the egg determined before fertilization and leading to asymmetrical divisions. Mammalian egg does not show any ...

Dorosh, Andriy; Margaryan, Hasmik; Vodička, Martin; Ergang, Peter; Šídová, Monika; Dvořáková-Hortová, Kateřina
Biotechnologický ústav, 2016

Distribution of CD46 and β1 integrin molecules with respect to different membrane structures of the sperm head
Šebková, Nataša; Frolíková, Michaela; Děd, Lukáš; Dvořáková-Hortová, Kateřina
2016 - English
CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction (AR). It also associates through membrane integrins with specific MAP kinases involved in the AR. Our aim was to monitor the dynamics of relocation of CD46 and β1 integrin during sperm maturation and its preparation for the fertilization. The dependence of this localization changes on the dynamic of actin cytoskeleton was studied. Our results show the changes in the localization of these proteins associated with the AR and their co-localization was observed using proximity ligation assay. After the AR CD46 and β1 integrin spreads across the sperm head, entering the post-acrosomal compartment, and permeates the borders of different domains. It was shown previously that actin dynamics is necessary for acrosome reaction-associated translocation of Izumo1 protein that is required for sperm-egg fusion. Therefore Latrunculin A was used during sperm incubation. The co-incubation of capacitated sperm with Latrunculin A leads to a decrease of the percentages of sperm, which express relocation pattern after induced AR. 3D models and visualizations of potential membrane processes responsible for the relocation of proteins from the acrosomal area to the other compartments of the sperm head were prepared. Our results deliver new information that proteins CD46 and β1 integrin undergo dynamic relocation towards the sites of sperm-egg fusion during the AR in vitro. The inhibitor of actin dynamics abrogates significantly the AR-associated changes in proteins localization. We speculate that this relocation is of importance for the successful sperm-egg interaction, adhesion and subsequent gamete fusion. Keywords: CD46; β1 integrin; acrosome reaction; latrunculin A; sperm-egg fussion; Proximity ligation assay Available at various institutes of the ASCR
Distribution of CD46 and β1 integrin molecules with respect to different membrane structures of the sperm head

CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction (AR). It ...

Šebková, Nataša; Frolíková, Michaela; Děd, Lukáš; Dvořáková-Hortová, Kateřina
Biotechnologický ústav, 2016

Super-resolution Stimulated Emission Depletion (STED) microscopy imaging of selected integrins in mouse acrosome intact sperm
Frolíková, Michaela; Šebková, Nataša; Děd, Lukáš; Dvořáková-Hortová, Kateřina
2016 - English
Keywords: actin; integrins family; STED; protein relocalization; acrosome membrane Available at various institutes of the ASCR
Super-resolution Stimulated Emission Depletion (STED) microscopy imaging of selected integrins in mouse acrosome intact sperm

Frolíková, Michaela; Šebková, Nataša; Děd, Lukáš; Dvořáková-Hortová, Kateřina
Biotechnologický ústav, 2016

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