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Influence of lamin A/C on paternal pronucleus DNA demethylation
Václavková, Vendula ; Fulková, Helena (advisor) ; Žatecká, Eva (referee)
A series of dynamic epigenetic changes need to happen to rebuild the gamete's genome after fertilization and to secure the totipotency of the zygote. The processes can be observed in both parental pronuclei, although the machinery providing the epigenetic dynamics probably lies in the maternal oocyte, more specifically in the germinal vesicle (GV). GV can be divided into two fractions: a soluble and an insoluble fraction. The probable ability of the soluble fraction is remodelling the sperm head and the paternal pronucleus formation, while the insoluble fraction could be involved in reprogramming of the paternal genome and ensuring the developmental competence. This thesis was focused on one of the epigenetic regulations which was DNA methylation and its rapid loss after fertilization. Putative mechanisms, which can occur there are the oxidation of methylated bases and their subsequent repair and replacement through the base excision repair (BER). This thesis tested a hypothesis that a protein lamin A/C, found in the insoluble fraction of the GV, is a possible candidate participating in the active demethylation through BER. To examine the role of lamin A/C in BER, it was required to prepare such GV oocytes, which would contain both the soluble part of GV and protein lamin A / C. The selective...
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Influence of tetrabromobiphenol A (TBBPA) on reproduction parameters, spermatogenesis and expression of selected genes in mice in vivo.
Žatecká, Eva ; Pěknicová, Jana (advisor) ; Kovář, Jan (referee)
Tetrabromobisphenol A is a substance that is widely used in industry as a fire retardant and is currently among the most widely used; its consumption is 210,000 tons / year and is still growing. It was shown that TBBPA can be released into the environment and it was found even in the human body. TBBPA has negative effects on different cell lines in vitro, including TM4 Sertoli cells. It was shown that it can affect the thyroid hormone system as well it might be able to influence the androgen / estrogen hormone system. Due to these facts, TBBPA was added to the list of potentially dangerous substances to human health. In this study, the effect of TBBPA on the reproductive parameters, spermatogenesis and activity of selected genes in mice in vivo was examined. First, we perform two generation in vivo study with outbred mouse line - CD1. In experimental animals we detected increased number of apoptotic cells in the testes, and also we observed changes in the expression pattern of selected genes encoding proteins that play an important role during spermatogenesis. In contrast, sperm quality and reproduction was not affected by TBBPA. As a complementary study was perform one generational in vivo study with inbred mouse line - C57BL/6. This study mostly confirmed the results obtained from previous in...
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Effect of selected endocrine disruptors on the male mouse reproductive system in vivo
Žatecká, Eva ; Pěknicová, Jana (advisor) ; Kubelka, Michal (referee) ; Kaňka, Jiří (referee)
In our environment there are many compounds which can negatively influence humans and wildlife. Every day, a vast number of environmental pollutants are released into our environment and there is no way to avoid their exposure. Some of these compounds can even mimic endogenous hormones and interfere with our endocrine system (so called endocrine disruptors), which is the key regulatory system controlling almost all physiological processes in human and animal bodies. Also the reproductive system is largely regulated by various hormones, and their proper function is crucial for gamete formation, fertilization and embryo development. Environmental pollutants are therefore considered as one of the possible causes of increased infertility in human population. This prompted us to study the effect of two endocrine disruptors (tetrabromobisphenol A - TBBPA, and zearalenone - ZEA) on the male mouse reproductive system in vivo. According to our results, TBBPA is able to induce apoptosis as well as changes in the expression of selected testicular genes and sperm protamination. Our results also suggest that permanent exposure to TBBPA slightly enhances its effect in the next generation, depending on whether the parents have been affected or not. We hypothesized that differential protamination of the sperm DNA...
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Influence of tetrabromobiphenol A (TBBPA) on reproduction parameters, spermatogenesis and expression of selected genes in mice in vivo.
Žatecká, Eva ; Pěknicová, Jana (advisor) ; Kovář, Jan (referee)
Tetrabromobisphenol A is a substance that is widely used in industry as a fire retardant and is currently among the most widely used; its consumption is 210,000 tons / year and is still growing. It was shown that TBBPA can be released into the environment and it was found even in the human body. TBBPA has negative effects on different cell lines in vitro, including TM4 Sertoli cells. It was shown that it can affect the thyroid hormone system as well it might be able to influence the androgen / estrogen hormone system. Due to these facts, TBBPA was added to the list of potentially dangerous substances to human health. In this study, the effect of TBBPA on the reproductive parameters, spermatogenesis and activity of selected genes in mice in vivo was examined. First, we perform two generation in vivo study with outbred mouse line - CD1. In experimental animals we detected increased number of apoptotic cells in the testes, and also we observed changes in the expression pattern of selected genes encoding proteins that play an important role during spermatogenesis. In contrast, sperm quality and reproduction was not affected by TBBPA. As a complementary study was perform one generational in vivo study with inbred mouse line - C57BL/6. This study mostly confirmed the results obtained from previous in...
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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
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
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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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Changes in the expression of selected testicular genes in mice
Valášková, Eliška ; Margaryan, Hasmik ; Žatecká, Eva ; Pěknicová, Jana
The decrease in population fertility has become a major concern in many developed countries. Recent studies show that infertility is affecting an estimated 15% of all couples (World Health Organization, WHO, 2010). Male infertility is the primary or contributing cause in 60% of cases. Male infertility is caused by a number of factors, such as genetic background, various environmental factors and disease. Diabetes mellitus (DM), a serious health problem on its own, is also suspected to be a contributing factor to male infertility. The aim of this project was to analyze the cellular, molecular and genetic effects of diabetic environment on spermatogenesis and sperm quality and to determine the impact of DM on the in vivo reproduction, using the mouse model (Mus musculus) inbred FVB. Diabetes was induced using streptozotocin. We used our knowledge and tools (unique monoclonal antibodies developed by our group) to determine the status of reproductive organs, anogenital distance, and the quality of sperms. Genetic analysis was performed by a quantitative Reverse Transcription Polymerase Chain Reaction (qPCR). We tested selected genes which are expressed in testicular tissue and thus can influence process of spermatogenesis and consequently the sperm quality. Our preliminary data strongly suggest that DM impairs male fertility. We have found significant changes in the body and reproductive organ weight of mice with DM. We have identified qualitative and quantitative changes in the expression of proteins in epididymal fluid and sperms. We have also detected an increased number of apoptotic cells in sperm of diabetic mice compared to the control group. To our knowledge, there is no study assessing the correlation between DM and “unexplained infertility”. In view of this, it is essential to analyze the effects of DM on male fertility, sperm quality, and reproduction parameters.
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