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The role of microRNAs in regulation of mammalian oocyte and embryo development
Marcollová, Kateřina ; Procházka, Radek (advisor) ; Svoboda, Petr (referee)
Cumulus-oocyte complex (COCs) is crucial for mammalian reproduction. Cumulus cells not only nurture the oocyte; they also represent important communicational nodes for mediating information towards and from the oocyte. The non-coding miRNAs can modulate posttranscriptional events they might serve as a useful biomarker for evaluating cell conditions. Based on the COCs staining with vital Lissamine Green B stain we divided cumulus cells into high- and low-quality ones. Furthermore, we implemented division based on the maturation stage, the GV and MII. Sequencing data analysis showed that DE miRNAs from qualitatively different stages donot significantly vary. Nonetheless, significantly DE miRNAs were detected between two developmentally different stages. We identified e.g. ssc-miR-183, ssc-miR-182, and ssc-miR-21-5p to be highly downregulated when comparing GV to MII stage. Among the highly expressed miRNAs from all samples were members of let-7 family (let-7c, let-7a, let- 7f-5p), ssc-miR-16, ssc-miR-21-5p, and ssc-miR-125a. Targeted genes by the DE miRNAs were involved in ErbB, TGF-β, MAPK, FoxO, gap junction and cGMP signalling pathways. We conclude that single miRNAs in cumulus cells probably cannot be used as a reliable oocyte quality marker. On the other hand,changes in the miRNA expression in...
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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NAD+-dependent histone deacetylase SIRT1 in the process of oogenesis, fertilization and early embryonic development
Valentová, Iveta ; Nevoral, Jan (advisor) ; Drutovič, David (referee)
SIRT1 is a histone deacetylase from the sirtuin family that affects epigenetic and non- epigenetic targets. We can assume that the known SIRT1 substrates are involved in the regulation of gametogenesis and early embryonic development. Our hypotheses say SIRT1 is present in oocytes and early embryos and it plays a physiological role in oocyte maturation, fertilization and early embryonic development. A mouse model of a conditional knock-out line producing Sirt1-deficient oocytes was developed to verify our hypotheses. Oocytes and embryos were analyzed for SIRT1, its selected substrates and other markers by immunocytochemistry. We found out that the presence of SIRT1 contributes to oocyte quality through modulation of the chromatin histone code and stabilization of the spindle. Furthermore, the purely maternal origin of SIRT1 presents in both zygote pronuclei. Last but not least we discovered a significant effect of SIRT1 on early embryonic development, probably mainly due to its role in the activation of the embryonic genome. The results confirm our hypothesis that SIRT1 is present in oocytes and embryos mainly around chromatin. The results show that SIRT1 is a maternal factor determining oocyte quality and it is necessary for the embryonic genome activation.
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Non-coding RNAs in oocyte and early embryo
Aleshkina, Daria ; Šušor, Andrej (advisor) ; Staněk, David (referee) ; Krylov, Vladimír (referee)
Once considered as 'transcriptional noise' noncoding RNAs (ncRNAs) nowadays are known to be key molecules in major cellular processes. NcRNAs are expressed at very high levels as only 2% of transcribed genome corresponds to protein-coding RNAs in higher eukaryotes. Various ncRNAs are known to have structural, functional, or regulatory roles, but the influence of the majority of non-coding transcripts is still unclear. Among ncRNAs, long ncRNAs (lncRNAs, longer than 200 bp) are of particular interest. LncRNAs do not have a uniform function but many studies observed lncRNA-based regulations at the transcriptional and translational levels. Therefore, novel lncRNAs could specifically fine-tune protein synthesis in the highly differentiated cell types. Particularly, fully-grown mammalian oocyte and early embryo require precisely controlled translation of maternal transcripts to coordinate meiotic progression and early embryo development while transcription is silent. We aimed to study the involvement of ncRNAs in protein synthesis and consequent influence on the oocyte and early embryo physiology. For the first time, we analysed the expression and distribution of several ncRNAs, namely Brain cytoplasmic RNA 1 (BC1), lncRNA in Oocyte Specifically Expressed (Rose), RNA Component of 7SK Nuclear...
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Effect of on in vitro maturation of porcine oocytes
Laštůvková, Viktorie ; Petr, Jaroslav (advisor) ; Benc, Michal (referee)
Lysmeral (Lilial, Butylphenyl Methylpropional) is a synthetically produced aldehyde. It is used for its typical lily of the valley-like scent as a perfume ingredient in cosmetic products. It is typically found, for example, in cleaning products. It is on the list of the 26 most well-known synthetic allergens and has been shown to be present in human urine and subsequently in wastewater. According to some opinions, lysmeral should be classified as an endocrine disruptor, which are substances that can interfere with the body's endocrine system. As these substances often affect mammalian reproduction, it is in society's interest to address them, given that they are commonly found in the environment. Lysmeral was not properly tested before being placed on the market and all its effects on organisms are still unknown. The hypothesis of this thesis is that exposure to lysmeral causes undesirable changes in the meiotic maturation of porcine oocytes in vitro and the aim is to investigate the effect of lysmeral, at concentrations corresponding to normal human exposures, on specific markers of meiotic maturation of porcine oocytes. The results confirm the negative effect of lysmeral on the smooth progression of meiotic maturation and the achievement of the final stage of porcine oocyte maturation in vitro....
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Study of changes on spindle and chromosomes vitrified and in vitro maturated human oocytes
Holubová, Kateřina ; Kratochvílová, Irena (advisor) ; Libusová, Lenka (referee)
The aim of this work is the comparison of cryoprotection methods for human oocytes regarding their survival rate and subsequent success in in vitro fertilization. This work showed the effect of oocyte maturation characterized by the state of the meiotic spindle, chromosomes and polar body on the success of in vitro fertilization of standard (fresh) and vitrified oocytes. The acquired knowledge will lead to a better understanding of the relationships between the state of oocytes and specific in vitro fertilization and cryoprotection procedures. The final, application-interesting goal will be to propose the optimization of methods for working with oocytes so that the probability of their success in the IVF cycle is as high as possible.
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MicroRNA pathway activity in mammalian oocytes
Kataruka, Shubhangini ; Svoboda, Petr (advisor) ; Simard, Martin (referee) ; Cochella, Luisa (referee)
(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events where a differentiated oocyte gives rise to a totipotent zygote. During the growth phase an oocyte prepares for fertilization and progression to zygotic genome activation. It does so by transcribing and storing the necessary mRNAs till a fully-grown oocyte attains transcriptional quiescence. Therefore, transcriptome regulation in a fully-grown oocyte is of utmost importance. Study of post-transcriptional regulatory pathways revealed that the small-RNA mediated regulatory pathways exist in a unique conformation in mouse oocytes. Endogenous RNAi pathway is essential for mouse female germline while miRNA pathway which is ubiquitously present in most cell types is dispensable for oocyte maturation and fertilization. My PhD project was aimed at understanding the constraints of the miRNA pathway in the oocyte which makes it non-functional. As a fully-grown oocyte is a huge cell with a proportionally large maternal transcriptome we analysed the miRNA: mRNA stoichiometry changes that occur from growing to the fully-grown mouse oocyte. Inability of miRNAs to accumulate during oocyte growth phase leads to their dilution in fully-grown oocyte rendering them inactive. Low miRNA concentrations were also observed in rat,...
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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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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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Regulation of mTOR pathway in the oocyte meiosis
Schmidtová, Barbara ; Šušor, Andrej (advisor) ; Krylov, Vladimír (referee)
Mammalian Target of Rapamycin (mTOR) is serin-threonin kinase, which has become a major topic in many studies in the last decade, leading to new insights into how cell works. This kinase is involved in proteosynthesis, metabolism, cell cycle regulation, proliferation and responses to nutrients and growth factors. There are certain diseases caused by mutations in mTOR gene, which lead to abnormal function of this kinase. These diseases include cancer or fertility disorders. mTOR research is also beneficial due to the search for drugs that could rescue its function and thus provide treatment for these diseases. The best-known drug is inhibitor Rapamycin and its derivates. The aim of this bachelor thesis is to summarize the knowledge about how mTOR can be regulated, the role of its substrates in cell function and to define the role of mTOR in oocyte development, translation and human health. Keywords: Oocyte, mTOR, translation, 4E-BP1, MPF, meiosis, AKT
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