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

Visualisation of CD46 and β1 integrins in mouse sperm head by super-resolution Stimulated Emission Depletion (STED) microscopy
Frolíková, Michaela; Šebková, Nataša; Děd, Lukáš; Dvořáková-Hortová, Kateřina
2015 - English Keywords: CD46 monoclonal antibody; β1 integrins; STED; Proximity ligation assay Available at various institutes of the ASCR Visualisation of CD46 and β1 integrins in mouse sperm head by super-resolution Stimulated Emission Depletion (STED) microscopy

Frolíková, Michaela; Šebková, Nataša; Děd, Lukáš; Dvořáková-Hortová, Kateřina
Biotechnologický ústav, 2015

CD46 and β1integrin interaction in mouse sperm head
Šebková, Nataša; Frolíková, Michaela; Dvořáková-Hortová, Kateřina
2015 - 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. Integrins interact with many cytoskeletal proteins such as actin, therefore changes in the actin cytoskeleton before and after AR may lead to changes in the association and localization of CD46 and β1integrin. Our aim was to monitor mutual CD46 and β1integrin interaction detected by the proximity ligation assay. It generates a localized signal in a form of spots revealing the exact position of the recognition event. Proteins interaction was study in freshly released sperm and sperm during the acrosome reaction, during which there is a gradual relocation of these proteins towards the equatorial segment and the whole sperm head. Proteins α and β tubulin were used as a positive control, α tubulin and β1 integrin as a negative control. In situ PLA showed a distinct spotted signal indicating the mutual interaction of CD46 and β1integrin. A positive response was demonstrated not only in freshly released sperm but also in sperm during the acrosome reaction. Freshly released sperm were distinctively labelled in the acrosome region and the neck, similarly to the positive control. Sperm during the acrosome reaction showed the signal across the whole sperm head region. No signal or sporadic nonspecific staining was detected in the case of the negative control. In summary, our results deliver new information that proteins CD46 and β1 integrin interact with each other. These results suppose the theory that β1 integrin can mediate a connection between CD46 and sperm cytoskeleton thereby molecules of signalling pathways leading to activation of the acrosome reaction. Keywords: CD46; β1 integrin; acrosome reaction Available at various institutes of the ASCR CD46 and β1integrin interaction in mouse 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. ...

Šebková, Nataša; Frolíková, Michaela; Dvořáková-Hortová, Kateřina
Biotechnologický ústav, 2015

Possible role of spermatogenic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) in mammalian sperm
Margaryan, Hasmik; Dorosh, Andriy; Čapková, Jana; Postlerová, Pavla; Philimonenko, Anatoly; Hozák, Pavel; Pěknicová, Jana
2015 - English Sperm proteins are important for the structure and function of these specific, highly differentiated cells. Certain of these proteins play a role in sperm-egg recognition during primary or secondary binding at zona pellucida glycoprotein matrix. The aim of this study was to characterize the acrosomal sperm protein recognized by a monoclonal antibody (MoAb) Hs-8, prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperm, and to test the possible role of this protein in gamete interaction. MoAb Hs-8 specifically labelled a 45 kDa protein from the sperm extract in the immunoblotting test. Sequence analysis identified this Hs-8 protein as GAPDHS. In order to perform a control tests, a commercial mouse anti-GAPDHS MoAb was applied. Both antibodies showed similar staining patterns using immunofluorescence labelling, transmission electron microscopy and immunoblot analysis. Moreover, both Hs-8 and commercial anti-GAPDHS antibodies blocked the secondary sperm-zona pellucida binding. Generally, GAPDHS was considered mainly as sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility and its role in the sperm head was unknown. In this study, we confirmed the potential additional role of GAPDHS as a binding protein that is involved in the sperm-zona pellucida interaction. Keywords: GAPDHS; sperm glycolytic enzyme; zona pellucida glycoprotein matrix; monoclonal antibody Available at various institutes of the ASCR Possible role of spermatogenic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) in mammalian sperm

Sperm proteins are important for the structure and function of these specific, highly differentiated cells. Certain of these proteins play a role in sperm-egg recognition during primary or secondary ...

Margaryan, Hasmik; Dorosh, Andriy; Čapková, Jana; Postlerová, Pavla; Philimonenko, Anatoly; Hozák, Pavel; Pěknicová, Jana
Biotechnologický ústav, 2015

Book of abstract XXIst Symposium of biology and immunology of reproduction
Pěknicová, Jana; Kubátová, Alena; Elzeinová, Fatima
2015 - English The Symposium was focused on immunology of reproduction and specific problems in reproduction (mainly in human infertility). Keywords: reproduction; infertility; capacitation; spermatozoa; anti-Mullerian hormone; oocytes; seminal plasma Available at various institutes of the ASCR Book of abstract XXIst Symposium of biology and immunology of reproduction

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

Pěknicová, Jana; Kubátová, Alena; Elzeinová, Fatima
Biotechnologický ústav, 2015

Sperm protein profiles of different mammalian species
Pohlová, Alžběta; Zigo, Michal; Jonáková, Věra; Postlerová, Pavla
2015 - English 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. Keywords: SDS-electrophoresis; sperm proteins; zona pellucida binding activity; monoclonal antibody Available at various institutes of the ASCR Sperm protein profiles of different mammalian species

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 ...

Pohlová, Alžběta; Zigo, Michal; Jonáková, Věra; Postlerová, Pavla
Biotechnologický ústav, 2015

Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla; Zigo, Michal; Pohlová, Alžběta; Jonáková, Věra
2015 - English 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: HPLC; lectins; zona pellucida glycoproteins; monoclonal antibody Available at various institutes of the ASCR Biochemical methods as tool for study of reproductive proteins

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 ...

Postlerová, Pavla; Zigo, Michal; Pohlová, Alžběta; Jonáková, Věra
Biotechnologický ústav, 2015

Diabetes mellitus negatively affects male reproductive parameters in vivo
Valášková, Eliška; Žatecká, Eva; Pavlínková, Gabriela; Bohuslavová, Romana; Dorosh, Andriy; Elzeinová, Fatima; Kubátová, Alena; Margaryan, Hasmik; Pěknicová, Jana
2015 - English According to the World Health Organization (WHO), 15% of couples in reproductive age suffer from infertility problems, and up to 60% of cases are caused by male factor. This could be caused by genetic background, environmental factors and various diseases, including diabetes mellitus (DM). However, the impact of DM on male fertility is not fully understood. . The aim of this study was to investigate the effects of DM on reproductive parameters and sperm quality, using mouse model. DM (type 1) was induced by Streptozotocin in FVB inbred mouse strain. Mice with blood sugar levels higher than 13.9 mmol/L were considered diabetic. After 4 weeks of diabetes exposure, diabetic males were bred with wild type females and transgenerational effect of DM was assessed. Selected morphological, cellular, and molecular parameters of diabetic males and their male offspring were compared to appropriate controls. There was an increased in sperm fragmentation and abnormalities of sperm morphology in diabetic mice in both generations. An increased staining with apoptotic marker annexin V was also detected in the diabetic groups. Furthermore, a presence of protamines as major sperm nuclear proteins was analysed. Protamine 1 to protamine 2 ratio (P1/P2), a marker of male fertility, was altered in sperms of experimental diabetic animals in both generations. Our findings indicate that DM type 1 negatively affects sperm quality and P1/P2 ratio and this negative effect is transmitted to the progeny Keywords: diabetes melitus; Sterptozotocin; apoptosis; protamination Available at various institutes of the ASCR Diabetes mellitus negatively affects male reproductive parameters in vivo

According to the World Health Organization (WHO), 15% of couples in reproductive age suffer from infertility problems, and up to 60% of cases are caused by male factor. This could be caused by genetic ...

Valášková, Eliška; Žatecká, Eva; Pavlínková, Gabriela; Bohuslavová, Romana; Dorosh, Andriy; Elzeinová, Fatima; Kubátová, Alena; Margaryan, Hasmik; Pěknicová, Jana
Biotechnologický ústav, 2015

Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Děd, Lukáš; Čapková, Jana; Kubátová, Alena; Teplá, O.; Pěknicová, Jana
2015 - English Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between normozoospermic and asthenozoospermic sperm samples. Since these studies were primarily focused on the identification of differentially expressed proteins by mass spectrometry, we aimed to evaluate the ability of our diagnostic antibodies to detect the differential expression of selected protein markers by fluorescent microscopy and flow cytometry techniques. Therefore, we analyzed sperm samples from 30 men with normal and 30 men with astheno spermiograms, average by the panel of our diagnostic anti-human sperm (Hs) antibodies. Fluorescent microscopy and flow cytometry analysis revealed quantitative differences in the protein abundances between normo and astheno sperm samples, namely, in GAPDHs, evaluated with Hs-8 MoAb, VCP, evaluated with Hs-14 MoAb, and ATP synthase, evaluated with MoAb Hs-36. From the methodological point of view, we observed very high correlation between the data obtained by fluorescent microscopy and flow cytometry techniques and therefore both methods are useful for evaluation of protein differences associated with asthenozoospermia. From the clinical point of view, we observed the strong association of the low sperm motility in the sample with the expression of proteins, playing an important role in sperm energy metabolism (expected), but also with the expression of all tested intra-acrosomal proteins. These findings further demonstrate asthenozoospermia as a complex semen disorder frequently associated with other semen pathologies, which are not diagnosed by basic semen analysis, and the possibility to use monoclonal antibodies as a tool for diagnosis of protein associated sperm pathologies in the semen with the low sperm motility. Keywords: monoclonal antibody; asthenozoospermia; flow cytometry Available at various institutes of the ASCR Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples

Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between ...

Děd, Lukáš; Čapková, Jana; Kubátová, Alena; Teplá, O.; Pěknicová, Jana
Biotechnologický ústav, 2015