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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
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.
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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.
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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
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.
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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
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.
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CD46 and β1integrin interaction in mouse sperm head
Šebková, Nataša ; Frolíková, Michaela ; Dvořáková-Hortová, Kateřina
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.
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Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina ; Frolíková, Michaela ; Děd, Lukáš ; Šebková, Nataša
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo in the female reproductive tract in order to obtain the ability to penetrate and fertilize the egg. CD46 and β1-integrin belong to specific proteins, which are predicted to interact during molecular reorganization of capacitating sperm. The IZUMO1 as the primary fusion protein of the mammalian sperm is also involved in this dynamic network. We investigated the relationship between the Izumo, CD46 and β1 integrin relocation in the sperm head during the capacitation and AR in vitro. We have already successfully monitored by immunofluorescent labelling the dynamics of proteins CD46 and β1-integrin. The changes in the localization of these proteins associated with the AR and their mutual co-localization was observed. The original β1-integrin location in the freshly released epididymal sperm is in the acrosome and it relocates during the AR further through the sperm head compartments into the equatorial segment and over the whole sperm head. Its density over the equatorial segment is decreasing with the extended time of the AR. Also its presence in the perforatorium of the mouse sperm head is very prominent. The pattern for protein CD46 is extremely similar if not identical in both aspects such as compartment localization and time progress during capacitation and AR in vitro. The molecular interaction of CD46 and β1-integrin was investigated using the Proximity Ligation Assay and Super resolution microscopy STED. The data were statistically analysed. The newly obtained results from CD46 and β1-integrin relocation are in correlation with IZUMO1 dynamics and giving a substantial knowledge on the studied protein network rearrangement during capacitation and AR in mouse spermatozoa.
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Patients satisfaction with the provided services. An important indicator of the quality of health care facilities.
MALANÍKOVÁ, Jana
My master's thesis which topic is "Satisfaction of patients with the services provided. An important indicator of quality of medical equipment " is mainly focused on satisfaction of patients with the services provided in the Hospital České Budějovice, a.s. Furthermore, the mapping focused on overall satisfaction with care in the already mentioned Hospital České Budějovice and on exploring whether patients evaluate the quality of information provided by a medical doctor in this hospital affirmatively and whether they are satisfied with care of local nurses. The satisfaction of patients is an important indicator of the quality of care provided, which has an impact on their status of basic human needs. One main purpose was determined: Monitoring the satisfaction of patients with the services provided in the Hospital České Budějovice, a.s. and this purpose was fulfilled. Quantitative research was executed, to its realization the interview method was used. By means of the technique of standardized anonymous questionnaires on the basis of the above-mentioned purpose,the 3 following hypothesis were verified: 1) Patients are satisfied with the overall care in the Hospital České Budějovice, a.s. 2) Interviewed patients evaluate the quality of information provided by a medical doctor in the Hospital České Budějovice, a.s. affirmatively 3) Interviewed patients are satisfied with the care of nurses in the Hospital České Budějovice, a.s. I processed the data to confirm or refute the pre-established hypotheses. The evaluation of the data was carried out using statistical nonparametric methods, Chi-square test and the chosen level of significance of p=0.05. The results of the research have been clearly processed into the tables and graphs. The program Microsoft Office Excel 2010 was used to process the results.
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Poliomyelitis and contemporary issues of its eradication
PETRÁŇOVÁ, Monika
This diploma thesis deals with the issue of the eradication of infectious polio and its current problems. Polio is a highly infectious disease of viral origin. The most effective form of protection from the disease is vaccination. The main aim of this work was to determine the major issues in polio surveillance in the Pilsen region at present. Intermediate goals were determined in relation to the main aim: to ascertain the awareness of practicing paediatricians about the principles of poliomyelitis surveillance with an emphasis on diagnosing and reporting cases of acute palsy and further to ascertain the quality of the cooperation between the locally competent public health authority and practicing paediatricians in the field of poliomyelitis surveillance. The work is divided into two parts, theoretical and practical. The practical part was elaborated using a qualitative method based on semi-structured interviews with practicing paediatricians and with the locally competent public health authority. The research was participated in by 9 practicing paediatricians and one public health authority worker from the Pilsen region, with an average length of practice of 20 years. The resulting data was then evaluated by a coding method and divided into schemes according to Švaříček and Šeďová (2007). Three research questions were defined based on the set aims: RQ1: What do practising paediatricians see as the prime problems in polio surveillance? RQ2: How many cases of acute palsy do practising paediatricians record and subsequently report to the competent public health authority? RQ3: What is the quality of the cooperation between practising paediatricians and the locally competent public health authority? The research revealed that, according to the practising paediatricians, the problem in poliomyelitis surveillance is refusal of the vaccination and the associated decreasing immunisation coverage of the population. The next most commonly reported problem was population migration. Furthermore, it was found that not one practising paediatrician recorded or reported cases of acute palsy in their surgery. Only one respondent encountered acute palsy 10 years ago. The answer to the last research question is also apparent from the information obtained. More than half of the respondents agreed that so far there is no cooperation on this issue. Although four respondents stated that the cooperation is at a high level and is of very good quality.
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Nové postupy biomonitoringu cizorodých látek ve vodním prostředí
ČERVENÝ, Daniel
Within the presented work, several approaches of biomonitoring were studied. A well-established method for human health risk assessment was applied to evaluate the quality of fish from open waters in the Czech Republic. As this work is targeted on sport fishermen, who frequently consume their own catches, besides the publication in scientific journal, a brochure with results of this study was distributed via the Czech Fishing Union. Health risks for consumers related to the consumption of wild fish were found strongly species and locality dependent. As there is no risks related with the consumption of carp (Cyprinus carpio) at all investigated sites, frequent consumption of predatory fish should be avoided at some fishing grounds. The use of passive samplers has become more and more attractive in the last two decades. As these devices are able to mimic the biological uptake of chemicals, their potential for replacing fish as bioindicators in routine biomonitoring programmes is evident. In our study, the comparison between analysis of fish tissues and POCIS extracts from selected sampling sites confirms this idea about interchangeability of these indicators in the case of PFASs as target pollutants. Moreover, no metabolic transformation of contaminants present in water take place in passive samplers contrary to living organisms, thus these reflect the actual contamination at the locality more precisely. The use of passive samplers completely fulfils the internationally accepted principles of Replacement, Reduction, Refinement (the three R´s). Another promising approach using YOY fish as bioindicators was evaluated within the present study. Multispecies samples of YOY fish homogenate was found as a better indicator for wide range of pollutants than muscle tissue of adult fish within one species from the same locality. As all inner organs and tissues are presented in analysed samples, more target pollutants in higher concentrations are detected compared to the muscle of adults. Besides the higher sensitivity in pollutants detection and quantification, practical and economic benefits are of concern in the case of the YOY fish approach. The YOY fish approach also particularly fulfil the three R´s strategy, as sampling of this age group of fish has a smaller impact on the environment than the sampling of adults. In case of mercury, that was found as a limiting contaminant for the consumption of fish from open waters in the Czech Republic, a nonlethal method of finclips sampling can be used for the monitoring of this compound in aquatic environments. Besides the monitoring, our optimised method for the estimation of muscle tissue concentrations could be used for human health risk assessment as well. The difference between real muscle concentrations and concentrations estimated using our method differs less than 10% in most of the investigated localities. Benthic organisms are an important part of the food web in aquatic environments, but insufficient information about its contamination is available. In another study included in this thesis, first evidence about bioaccumulation of certain pharmaceuticals in benthic organisms was described. Although, pharmaceuticals are generally considered to be not accumulative in organisms, our study proved that certain pharmaceuticals have bioaccumulation potential. From this finding, it is evident that not only the concentrations of pharmaceutical dissolved in water but also the diet represents an important exposure pathway of contamination by these compounds for organisms (fish) inhabiting aquatic environments.
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Political aspects of local government – elections and voting behaviour of inhabitants of municipalities of Blovice city
Jan, Jaroslav ; Kopřiva, Radek (advisor)
Despite the transformation of party systems both at the national and sub-national political decision-making levels of politics, the hallmark of local political life of the municipality of Blovice city is a relative stability of the local party system. This fact is the basis of the Master thesis which aims to assess the nature of the electoral behavior of the local electorate in the elections to the municipal council with the ambition to evaluate the utility of the theory that is based on locally contextual interpretation. Partial objectives of the thesis that contribute to the main goal are the following: a description of the results of elections that are decisive for a development of the party system of Blovice city, a description of variables that underlie the permanent electoral support for the Civic Democratic Party (ODS) and the characteristics of electoral behavior of the local electorate. Thesis will be designed as a case study. A research will be based on voting decisions of electorate of Blovice city. The objects of the research will be the voters who participate in the elections to the municipal council and representatives of local politics and territorial autonomy. Value attitudes and voting behavior of voters will be assessed on the basis of data obtained by questionnaire. Electoral support of parties will be evaluated on the basis of data from unstructured interviews with local politicians (especially those from the party ODS). A local political life and in particular the election results will be described thanks to data published by the Czech Statistical Office (available on www.volby.cz).
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