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
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 ...
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 ...
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
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 ...
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 ...
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
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. ...
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 ...
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 ...
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