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The role of SIRT1 during in vitro maturation of oocytes
Landsmann, Lukáš ; Nevoral, Jan (advisor) ; Šolc, Petr (referee)
SIRT1 histone deacetylase acts towards many epigenetic and non-epigenetic targets. The involvement of SIRT1 in oocyte maturation is assumed and the importance of ooplasmic SIRT1 pool for further destiny of matured oocyte is strongly suggested. We hypothesized that SIRT1 play role of the signal molecule in mature oocyte through selected epigenetic and non- epigenetic regulation. We observed SIRT1 re-localization in mature oocyte and the association with spindle microtubules. In matured oocyte, SIRT1 shows a spindle-like pattern and spindle- specific SIRT1 action is supported decreasing α-tubulin acetylation. Based on the observation of histone code in immature and matured oocytes, we suggest that SIRT1 is mostly predestined for epigenetic mode of action in germinal vesicle (GV) of immature oocyte. Accordingly, SIRT1- driven trimethylation of histone H3 on lysine K9 in matured oocyte is considered to be an inheritance of GV epigenetic transformation. Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocyte capable to be switched from epigenetic to the non- epigenetic mode of action readily depending on meiosis progress. Keywords: oocyte, SIRT1, histone, developmental competence, tubuline, epigenetics
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Visualization of RNA and its translation in mammalian oocyte
Bašus, Kryštof ; Tětková, Anna (advisor) ; Macůrková, Marie (referee)
Translation is one of the fundamental biological processes. It is assumed that the locali- zation of mRNA and its translation in a certain time and at specific locations plays a role in many cellular processes. The mammalian oocyte becomes transcriptionally inactive when it reaches its full size and utilizes only RNA that has been synthesized and stored in the early stages of development. Thus the regulation of protein synthesis at the translational level is cri- tical for the correct completion of meiotic maturation of oocytes and early development of embryos. To monitor cellular physiology, it is necessary to be able to visualize and monitor spe- cific molecules and processes at single cell level. This is enabled by the development of light microscopy and fluorescence probes that specifically bind to certain organelles, cellular struc- tures, proteins or other molecules. In this thesis I describe selected methods of visualization of RNA, global translation as well as translation of specific transcripts, and proteins. The methods that this thesis describes are RNA FISH, visualization of translation using methionin analogs, FUNCAT, SUnSET, FlAsH, ReAsH, TRICK, SINAPS, FUNCAT-PLA, PURO-PLA. Key words: Translation, RNA, Protein, Visualization, Oocyte
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Identification of key regulators of gene expression in mammalian oocyte and embryo
Jansová, Denisa ; Šušor, Andrej (advisor) ; Gahurová, Lenka (referee) ; Lin, Chih-Jen Lance (referee)
Mammalian oocyte is a highly differentiated cell which gives rise to an embryo after fertilization. Importantly, fully-grown oocytes become transcriptionally inactive at the end of the growth phase. During following stages of development, i. e. meiotic maturation of the oocyte and early embryonic development, only transcripts previously synthesized and stored are used. The tight correlation between mRNA distribution and subsequent protein localization and function provides a mechanism of spatial and temporal regulation of gene expression used by various cell types. However, not much is known about mRNA localization and translation in the mammalian oocyte and early embryo. The aim of my thesis was to determine the localization of transcripts and components of translational machinery in the mammalian oocyte and embryo and to uncover the mechanisms of spatiotemporal regulation of translation as a prerequisite for correct oocyte and embryo development. We have shown that nuclei of both mouse and human oocytes contain RNA molecules and RNA binding proteins. Following the nuclear envelope breakdown (NEBD), translational hot-spots occur in the area surrounding the nuclear region. We suppose that mRNAs previously retained in the nucleus are released to the cytoplasm during NEBD and their subsequent...
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Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Flachsová, Monika
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
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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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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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Effect of hydrogen sulphide on aging of porcine oocytes in vitro
Krejčová, Tereza ; Petr, Jaroslav (advisor) ; Ješeta, Michal (referee)
Unfertilized or parthenogenetically non-activated porcine oocytes matured in vitro conditions are subjected to a process known as aging. During such development, porcine oocytes undergo the complex of the structural and functional changes, which can result in spontaneous parthenogenetic activation, fragmentation or lysis. After three days of culture in our condition, 23% of oocytes remained at the stage of metaphase II, 48% of oocytes were spontaneously parthenogenetically activated, 26% of oocytes were subjected to fragmentation and 3% of oocytes were lysed. The complete suppression of porcine oocyte fragmentation during the process of aging occurred during oocytes cultured in medium with sulphide donor Na2S in concentrations 150 µM and 300 µM. Inhibition of enzymes catalyzing the synthesis of hydrogen sulfide in the oocytes during the process of aging (cystathionine-gamma-lyase and cystathionine beta-synthase), iniciates earlier onset of oocytes fragmentation. The effect of both inhibitors could be completely reversed by using sulphide donor Na2S. The process of aging in porcine oocytes significantly reduces the success of the activation processes. Parthenogenetic activation occurred in 94% of pig oocytes, which were not subjected to aging. The proportion of activated oocytes after exposure to 24...
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Analysis of short Argonaute isoforms from mouse oocytes
Jankele, Radek ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...
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Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Šídová, Monika ; Tlapáková, Tereza (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
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Psychosocial problems of infertility verbaled by patients of fertility treatment ambulance.
DVOŘÁKOVÁ, Alena
Infertility is the inability to naturally conceive a child or to carry a pregnancy to full term. There are many reasons why a couple may not be able to conceive, or may not be able to conceive without medical assistance. Infertility may have profound psychological effects. Partners may become more anxious to conceive. Marital discord often develops in infertile couples, especially when they are under pressure to make medical decisions. Women trying to conceive often have clinical depression. Even couples undertaking IVF face considerable stress, especially the female partner. In vitro fertilisation (IVF) is a technique in which egg cells are fertilised by sperm outside the woman's womb. IVF is a major treatment in infertility when other methods of achieving conception have failed. The process involves hormonally controlling the ovulatory process, removing eggs from the woman's ovaries and letting sperm fertilise them in a fluid medium. The fertilised egg (zygote) is then transferred to the patient's uterus with the intent to establish a successful pregnancy. In this study ten couples using fertility treatment were analysed. The purpose of this study was to follow and describe their problems during the treatment, troubles with partners, jobs and friends. The author tried to find a solution how to communicate with patients, how to help them to manage the treatment. The elected experimental strategy was qualitative analysis. This study could be used by stuff in centres of artificial reproduction.
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