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Bioinformatic analysis of RNA dynamics during mammalian oocyte-to-embryo transition
Horvat, Filip ; Svoboda, Petr (advisor) ; Bruce, Alexander (referee) ; Vioristo, Sanna (referee)
The oocyte-to-embryo transition (OET) is a complex biological process during which a terminally differentiated oocyte undergoes numerous and coordinated changes to emerge as a collection of totipotent cells - initial blastomeres of a preimplantation embryo. In mammals, this process is primarily controlled through post-transcriptional regulation of maternal RNAs and transcriptional induction of zygotic RNAs. Technological advancements of next-generation sequencing methods during the last decade enabled studying OET through bioinformatic studies of high-throughput transcriptomics and genomics datasets. The work presented in this thesis explores mechanisms of post-transcriptional regulation and dynamics of different RNAs during OET by utilizing in-depth computational analyses. The presented work is divided into three topics, all covering distinct regulatory facets of the mammalian OET and illustrating roles of different RNA species. The first topic discusses dynamics of maternal transcriptomes in mouse. In our research, we explored roles of deadenylase CNOT6L in the maternal mRNA turnover in mice. We provided evidence that animals deficient in Cnot6l expression are subfertile, due to disruption in deadenylation and degradation of maternal mRNAs deposited in the oocyte. In another study, we...
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Endogenně produkovaný sulfan v reprodukčním traktu samice ve vztahu k fertilizaci
BRICHCÍN, Jiří
The discovery of the mechanisms responsible for the proper process of fertilization and early embryonic development helps in the development of assisted human reproduction and technologies in livestock breeding. One of the regulatory mechanisms involved in reproduction is the gasotransmitter hydrogen sulfide, whose cellular signaling in germ cells is not fully described yet. According to the hypothesis, hydrogen sulfide is released in the female reproductive tract, where it physiologically affects sperm and is necessary for fertilization and the development of pronuclei. The aim was to verify the expression of hydrogen sulfide-releasing enzymes and the existence of a time-space gradient of hydrogen sulfide within the estrous cycle of the mouse (time axis) and to compare the expression profile of the fallopian tube ampulla and uterotubal junction (space axis). The experiments were performed on a laboratory mouse model (Mus musculus). The expression of hydrogen sulfide-releasing enzymes was studied by western blot. Hydrogen sulfide production was determined by colorimetry. The role of hydrogen sulfide in the fertilization process was studied by in vitro fertilization with hydrogen sulfide donor-treated sperm and by immunocytochemical staining of the zygotes. The expression of the known hydrogen sulfide-releasing enzymes, i.e. cystathionine--lyase (CTH), cystathionine-ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MPST), was detected within the ovary and fallopian tube. Any enzymes did not show statistically significant differences in estrus and diestrus expression. Moreover, analogous results were found in the spatial axis; both the fallopian tube and the uterotubal junction expressed CTH, CBS, and 3-MPST enzymes, but no statistical differences were noted. Also, this applies to the production of hydrogen sulfide by these tissues. The effect of hydrogen sulfide on the fertilization process was analyzed according to lamin B1, a paternal pronucleus development marker. The performed analysis did not confirm the influence of the hydrogen sulfide donor on the development of the zygote pronuclei. The work showed that hydrogen sulfide is enzymatically released by the tissues of the female reproductive tract, regardless of the phase of the estrous cycle or the location in the fallopian tube. There is assumed a physiological influence of maternal-born hydrogen sulfide to sperm, which is capable of fertilization and leads to pronuclei development.
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Long Non-Coding RNAs in Oocyte-to-Embryo Transition
Ganesh, Sravya ; Svoboda, Petr (advisor) ; Vanáčová, Štěpánka (referee) ; Shkumatava, Alena (referee)
(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events, during which a differentiated oocyte gives rise to a totipotent zygote. During OET a transcriptionally silent oocyte undergoes massive reprogramming of gene expression, which transforms it into a transcriptionally active zygote. Although numerous studies have contributed to understanding the mechanism of OET, many genes involved in OET are yet to be identified. A whole new level of possible regulation of OET came with the discovery of long non-coding RNAs (lncRNA). LncRNAs are pol II transcripts longer than 200 nucleotides, that are typically spliced and polyadenylated but do not encode proteins. While lncRNAs have been studied in many model systems including embryonic stem cells, their expression in oocytes and early embryos and contribution to OET were largely unexplored at the beginning of this project. In my PhD project, I aimed to identify, annotate, and analyze lncRNAs expressed during OET. First, using RNA-Seq, 1600 highly reliable lncRNAs were identified and annotated in mouse oocytes and early embryos. Majority of lncRNAs were novel with expression exclusively at OET stages. A significant fraction of these lncRNAs was found associated with LTR retrotransposons, contributing to their novelty and...
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Analysis of pluripotent gene expression program in early embryos and embryonic stem cells
Moravec, Martin ; Svoboda, Petr (advisor) ; Motlík, Jan (referee)
Pluripotence je schopnost buňky diferencovat do jakéhokoliv buněčného typu. Formuje se během časného embryonálního vývoje u savců a její vznik je spojen s reprogramací genové exprese na globální úrovni. Proces přirozeného vzniku pluripotence není stále zcela pochopen. Pro získání nového pohledu na události, které vedou ke vzniku pluripotence u savců, studovali jsme změny v genové expresi během oocyt-zygotického přechodu u myši. V tomto modelovém systému, oplodněné vajíčko podstoupí reprogramaci, která vede k vytvoření pluripotentních blastomer. Tyto blastomery zakládají samotné embryo. Cílem mé diplomové práce bylo analyzovat aktivaci transkripce během časného vývoje a vyvinout metodu pro monitorování exprese genů v oocytech, časných embryích a embryonálních kmenových buňkách. Metoda využívá kvantitativní PCR a umožnuje změřit expresi až 48 vybraných genů, které slouží jako markery pro maternální degradaci, aktivaci pluripotentního programu a diferenciaci do zárodečných linií. Dále ukazujeme, že náš systém monitoruje dynamiku transkriptomu během oocyt-zygotického přechodu, a získané výsledky jsou srovnatelné s daty naměřenými pomocí jiných metod. Díky našemu bioinformatickému přístupu jsme navíc identifikovali nové oocyt-specifické a zygotické nekódující RNA. Klíčová slova: pluripotence,...
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Role of sirtuins in pronucleus formation in vitro fertilised porcine oocytes
Maryníková, Veronika ; Žalmanová, Tereza (advisor) ; Miriama , Miriama (referee)
Recently, the increasing importance of reproductive biotechnologies rises. They provide us to get higher performance of livestock or to improve treatment in human medicine. It is neccessary to have a sufficient amount of developmentaly competent oocytes and further healthy liveable embryos for in vitro culture to supply a progress of reproductive technologies. Immediately after fertilization, pronucleus formation is a key moment for
further embryonicdevelopment. Male and female pronuclei have their own pattern of histone code. For development of early embryo, it is neccessary to supply the correct pattern of histone code. NAD+-dependent histon deacetylases, sirtuins, are one of the mechanism which
plays in regulation of histone code. These family contains seven isoforms, SIRT1-7.
Based on current research, we decided for hypothesis that sirtuins are present in porcine fertilized oocytes and regulate the pronucleus formation. In this thesis, porcine COCs were culture in modificated culture medium and after 44 hr. maturation, only oocytes with extruded first polar body were chosen and used for further in vitro fertilization. Presumed zygotes were subsequently cultured with sirtuins inhibitors, nicotinamide or sirtinol. After 22 hr. of in vitro culture, zygotes were subjected by imunocytochemicaly localization of
methylated and acetylated (on lysine K9) histone H3 and image analysis.
Our results show that SIRT1 is localizated in porcine zygotes, especially in pronuclei. There are changes in acetylation and methylation H3K9 after sirtuin inhibition. Significant increase of H3K9 acetylation and decrese in H3K9 methylation are appeared. Sirtinol usage
has confirmed that the changes are result of SIRT1 action. Role of SIRT1 in histone code regulation of pronucleus formation is still not enought described in porcine.
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Histone code and its regulation during early embryonic development in pigs
Jelínková, Pavla ; Žalmanová, Tereza (advisor) ; Miriama, Miriama (referee)
Both pronuclei of the zygote undergo epigenetic changes after fertilization, which determines the quality of the zygote and successful early mammalian embryonic development. Shortly after fertilization epigenetic asymmetry among the pronuclei of the zygote is evident, while the paternal pronucleus undergoes active DNA demethylation, the DNA of the maternal pronucleus remains methylated. The male pronucleus in addition undergoes histone acetylation, whereas the histones of the female pronucleus remain methylated. Asymmetry of pronuclei and their epigenetic status predicts successful reprogramming of the genome, and thus the success of embryonic development. For the successful development of the embryo is therefore required correct formation of both of these pronuclei of the zygote and this formation of pronuclei is regulated by post-translational histone modifications called histone code. It was hypothesized that the histone code is regulated by the activity of NADP+ - dependent histone deacetylases, sirtuins.
In the experiment were used fully grown in vitro maturated pig oocytes that were fertilized with pig spermatozoa in vitro. After isolation of zygotes cultured with addition of the activator sirtuin resveratrol was performed immunofluorescence analysis of acetylated and methylated histone H3 at lysine K9 of pronuclei of the zygotes. From the results of control group asymmetry between the pronuclei of the zygote is evident; wherein the male pronucleus exhibits higher acetylation intensity contrast female pronucleus exhibits higher methylation intensity. After adding resveratrol to all experimental groups female pronucleus showed a significant increase of the methylated histone H3 at lysine K9, and contrary to the male pronucleus significant decrease of acetylated histone H3 at lysine K9.
Sirtuins are involved in the regulation of histone code in porcine zygote and it can be assumed that they also play a role during subsequent embryonic development, which is the subject of further study.
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