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Cell cycle regulation and genome integrity protection in the early mammalian embryos
Knoblochová, Lucie ; Drutovič, David (advisor) ; Carr, Antony M. (referee) ; Fulková, Helena (referee)
(English version) Infertility is a major health problem, as it affects one in every six people worldwide (Njagi et al., 2023). One of the major reasons for infertility are aneuploidies, additions or losses of an entire or partial chromosome during cell division. Aneuploidies thus negatively influence cellular processes and potentially lead to developmental problems or embryo loss. It has been thought for a long time that aneuploidies arise mostly during oocyte development, and these mechanisms have been well studied. However, recent evidence has shown that aneuploidies arise also de novo after fertilisation and during the early embryonic development; but the molecular mechanisms of these abnormalities still remains elusive. Aneuploidies often originate during cell cycle division from unrepaired DNA damage in mitosis. DNA damage is sensed by DNA damage response (DDR) signalling pathways, which slow down or arrest cell cycle progression until it is resolved. An essential DDR factor during typical cell cycle progression is checkpoint kinase 1 (CHK1). However, the role of DDR factors in the early embryos, and especially CHK1, have not been well studied. Early embryonic development is regulated by maternal factors stored in the oocyte until the transcription of the embryonic genome begins. To study...
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Bioinformatics analysis of sequences required for localization of RNA during development
Naraine, Ravindra ; Šindelka, Radek (advisor) ; Fulková, Helena (referee) ; Tichý, Boris (referee)
The development of a complex organism from the fusion of two cells (oocyte and sperm) has been a fascinating aspect of developmental biology. It is now known that certain spatially and temporally regulated molecules tightly regulate embryogenesis. The asymmetrical gradient of these molecules within a given cell or within groups of cells helps to guide the differentiation of certain parts of the developing embryo. In fishes and frogs, the establishment of the maternal animal-vegetal transcript gradient within the egg produces the first developmental axis and subsequent formation of the ectoderm, mesoderm, and endoderm regions. Despite this important process, most of our knowledge on this initial animal-vegetal distribution in vertebrates has been limited primarily to the Xenopus laevis model, involved the analysis of only few transcripts and also analyzed only polar regions of the oocyte. This thesis aims to address this deficit in knowledge by leveraging high throughput analysis (RNA sequencing) to characterize and compare the maternal transcriptome and its sub-compartmentalization within the egg of four distantly related models. Additionally, we analyzed different stages of oocyte maturation to determine where the observed localization occurs. The models used were the African clawed frog (Xenopus...
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The effects of epigenetic factor PRDM9 on the fertility of rodent females
Gašić, Srdan ; Trachtulec, Zdeněk (advisor) ; Děd, Lukáš (referee) ; Fulková, Helena (referee)
The Prdm9 gene encodes a histone-3-lysine-4,36-trimethyltransferase that specifies meiotic recombination sites and guides programmed double-strand breaks (DSBs) in mice, rats, and humans. Some vertebrates lost Prdm9 but not fertility throughout evolution, while the removal of Prdm9 caused sterility in some mouse strains, such as C57BL/6 (B6). The reasons for such species-specific fertility differences are unknown. To resolve these different requirements for PRDM9 in fertility, we produced Prdm9 mutants in another mammalian species, Rattus norvegicus, strain SHR/OlaIpcv. The removal of Prdm9 function did not completely abolish fertility in rats (as in B6 mice). Here I demonstrate that the loss of rat PRDM9 delayed female meiosis and caused synapsis and DSB repair defects that lead to a significant oocyte loss. However, unlike Prdm9-deficient B6 mouse oocytes, about 10-15% of pachytene-like mutant rat oocytes synapsed their chromosomes and repaired DSBs to the levels similar to controls. Because of this, female rats lacking PRDM9 maintained some oocytes until adulthood and yielded offspring, while B6 mice lost oocytes around the time of birth. Nevertheless, the adult rat mutant oocytes were exhausted earlier than the control adult rat oocytes. Therefore, PRDM9-lacking female rats suffered from...
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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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Manipulating the mammalian oocyte and embryo - Biological and epigenetic aspects
Fulková, Helena ; Hozák, Pavel (advisor) ; Hampl, Aleš (referee) ; Motlík, Jan (referee)
CONCLUSIONS . By antibodies against ďfferent covalent histone modifications and 5-methylcytosine, we have partialty characterised the epigenetic changes taking place during the oocyte mauration and in early mammalian embryogenesis in the mouse and pig, respectively. o We have also characterised thc epigenetic repogramming activities of cytoplasts derived from oocytes at different stages of maturation after somatic cell nuclear transfer. . We have evaluated the epigenetic effec$ of selected procedures that are currently used for embryo production. . Finally, we have developed a new cryopreservation scheme for oocyte nuclear material storage. orrr research is engaged in the development ofnew bíotechniques as well as elucidating and characterising the epigenetic pÍocesses that take place during normal and abnormal embryogenesis. Abnormal embryonic development is for example often observed in somatic cell nuclear transfer embryos. These techniques can also be potentially used not only in human medicine but also for valuable livestock and endangered species preservation Oy e.g. interspecies nuclear transfer). Especially in human meďcine, attention to the ethical issues associated with these techniques must be paid. It is also clear tbat many biological problems still do exist and these should not be...
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Porcine models for Huntington disease
Růna Vochozková, Petra ; Motlík, Jan (advisor) ; Bohačiaková, Dáša (referee) ; Fulková, Helena (referee)
The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...
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Porcine models for Huntington disease
Růna Vochozková, Petra ; Motlík, Jan (advisor) ; Bohačiaková, Dáša (referee) ; Fulková, Helena (referee)
The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...
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Regulation of translation in mammalian oocytes and early embryos
Jindrová, Anna ; Šušor, Andrej (advisor) ; Flemr, Matyáš (referee) ; Fulková, Helena (referee)
Fully grown oocytes undergo their further development in the absence of transcription. Completion of meiosis and early embryo development rely on the maternal mRNAs synthetized and stored during earlier development. Thus, the regulation of gene expression in oocytes during that period is controlled almost exclusively at the level of mRNA stabilization and translation. In the same vein, any mRNA metabolism could play a critical function at this stage of development. RNA localization followed by a local translation is a mechanism responsible for the control of spatial and temporal gene expression in the cell. We focused on visualization of mRNA and in situ translation in the mammalian oogenesis and embryogenesis. We characterized localization of global RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. Additionally we visualized specific ribosomal proteins that contribute to translation in the oocyte and embryo. We have shown that the key player of cap-dependent translation mTOR becomes highly active post nuclear envelope breakdown (NEBD) and in turn its substrate, translational repressor 4E-BP1 becomes inactive. Precise localization of inactivated 4E-BP1 at the newly forming spindle of the oocyte indicates the ongoing translation in this area. Furthermore, from...
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Manipulating the mammalian oocyte and embryo - Biological and epigenetic aspects
Fulková, Helena ; Hozák, Pavel (advisor) ; Hampl, Aleš (referee) ; Motlík, Jan (referee)
CONCLUSIONS . By antibodies against ďfferent covalent histone modifications and 5-methylcytosine, we have partialty characterised the epigenetic changes taking place during the oocyte mauration and in early mammalian embryogenesis in the mouse and pig, respectively. o We have also characterised thc epigenetic repogramming activities of cytoplasts derived from oocytes at different stages of maturation after somatic cell nuclear transfer. . We have evaluated the epigenetic effec$ of selected procedures that are currently used for embryo production. . Finally, we have developed a new cryopreservation scheme for oocyte nuclear material storage. orrr research is engaged in the development ofnew bíotechniques as well as elucidating and characterising the epigenetic pÍocesses that take place during normal and abnormal embryogenesis. Abnormal embryonic development is for example often observed in somatic cell nuclear transfer embryos. These techniques can also be potentially used not only in human medicine but also for valuable livestock and endangered species preservation Oy e.g. interspecies nuclear transfer). Especially in human meďcine, attention to the ethical issues associated with these techniques must be paid. It is also clear tbat many biological problems still do exist and these should not be...
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Generation and analysis of double deficient transgenic mice for kallikrein-related peptidase 5 and kallikrein-related peptidase 14
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Fulková, Helena (referee)
Kallikrein-related peptidases (KLKs) constitute a highly conserved serine protease family. Based on in vitro experiments, KLKs are predicted to play an important role in a number of physiolog- ical and pathophysiological processes. However, their role in vivo remains not fully understood, partially due to a lack of suitable animal models. In this work, we aim to prepare a KLK5 and KLK14 double-deficient mouse model. Both KLK5 and KLK14 were proposed to be involved in epidermal proteolytic networks critical for maintaining skin homeostasis. However, both KLK5 and KLK14 single-deficient mouse models show minimal or no phenotype, likely due to similar substrate specificity resulting in functional compensation. Double-deficient mice cannot be easily obtained by crossing due to localization of the Klk5 and Klk14 genes within the same locus on chromosome 7. We report that KLK5 and KLK14 double-deficient mice were success- fully generated, mediated by transcription activator-like effector nucleases (TALENs) targeting Klk14 by microinjection of TALEN mRNA into KLK5-deficient zygotes. Furthermore, we show that KLK5 and KLK14 double-deficient mice are viable and fertile. We believe that these novel mouse models may serve as a useful experimental tool to study KLK5 and KLK14 in vivo.
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