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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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Detection and quantification of maternal RNA localization during early development
Šimková, Kateřina ; Šindelka, Radek (advisor) ; Frolíková, Michaela (referee)
The asymmetric localization of maternal RNAs and proteins is a crucial mechanism for the body plan development in many animal species. These maternal factors are expressed during the oogenesis and they are used for the regulation of early developoment. In this diploma thesis, I addressed the role of asymmetrically localized RNAs along animal- vegetal axis in the early development of Ambystoma mexicanum. The second part of my thesis is focused on RNA localization in dorso-ventral and left-right axes. I identified the localization patterns of many known RNAs along the animal-vegetal axis using RT-qPCR and I also detected several genes, which can be involved in the dorso-ventral or lef-right patterning. Furthermore, we performed transcriptome wide analysis, which revealed changes classified into the following categories: RNAs relocalization, de novo synthesis before the onset of MBT and RNAs degradation during early development. Many vegetally localized genes in Xenopus laevis with the important role in the development of primordial germ cells have been previously described. Surprisingly, my results show that many of these genes are degraded during the early development of A. mexicnum. I believe that this degradation may be due to a different mechanism of PGC development in A. mexicanum and X. laevis....
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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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Regulation of development of mouse parthenogenetic embryos
Jettmarová, Dominika ; Fulka, Josef (advisor) ; Kaňka, Jiří (referee)
The development of mouse (Mus musculus) haploid parthenogenetic embryos does not reach the same level as normal embryos. The aim of this diploma thesis was to find out whether haploid parthenogenetic embryos of mice differ in the nucleocytoplasmic ratio. The volume of the nucleus increases with ploidity. The nucleocytoplasmic ratios of haploid embryos do not significantly change between the two-cell and four-cell stage (p = 0.052), there is a significant difference (p < 0.001) for diploid and tetraploid embryos. Non-standard nucleocytoplasmic ratio could be related to the problematic development. Understanding the regulation of preimplantational development of parthenogenetic embryos will increase the efficiency of haploid embryonic stem cell derivation.
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Gene expression in chicken embryo: micromanipulation and visualization methods
Bendová, Michaela ; Dvořák, Michal (advisor) ; Hirsch, Ivan (referee) ; Krylov, Vladimír (referee)
The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...
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Role of Nkx2.5 in development and electrophysiology of the mouse heart
Hámor, Peter ; Sedmera, David (advisor) ; Neckář, Jan (referee)
Role of Nkx2.5 in development and electrophysiology of the mouse heart Prague 2016 Peter Hámor, B.S. ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse hearts of different genotype. Special method of capturing the conduction of electric impulse through myocardium, called optical mapping, was used to visualize the electrical activity. Thanks to this method I was able to construct images and videos capturing the spread of the impulse with identification of the beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects in mutants compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Embryos at this developmental stage were optically mapped and analysed according to their genotype. While the wild type and heterozygote mouse embryos exhibited high degree of similarity, the homozygous mutants were dramatically different. Considering this work...
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Prenatal and perinatal loss entangled in Czech institutions
Hintnausová, Marie ; Zandlová, Markéta (advisor) ; Klepal, Jaroslav (referee)
PRENATAL AND PERINATAL LOSS ENTANGLED IN CZECH INSTITUTIONS Marie Hintnausová ABSTRACT An ethnographic research among women who experienced a loss of child during pregnancy or postpartum aims to expose contexts and backgrounds in which prenatal and perinatal loss emerges in the Czech Republic. This life event is delineated not only by unique biographies of affected mothers, but also by the societal understanding of prenatal life and institutional definitions of human reproduction. This thesis highlights various notions and meanings entangled in the event of prenatal and perinatal death and shows which trajectories women follow in the terrain of societal expectations and governmental and biomedical institutions when they lose a promised assurance of raising a new child.
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The legal status of the embryo from the perspective of European human rights protection
Havlíková, Barbora ; Pítrová, Lenka (referee)
Title The legal status of the embryo from the perspective of European human rights protection Abstract The thesis addresses the question regarding the legal status of the human embryo in European law. The aim of the thesis is to evaluate whether the embryo stands in the position of a legal object or legal subject and whether the legal status of embryo is in compliance with natural law. For this purpose the thesis firstly defines the status in the field of natural law, i.e. the moral status of embryo. Afterwards, the thesis examines the legal position of embryo in human rights protection. The thesis answers the question whether the embryo is subject of human rights documents. The legal status of embryo is analyzed in relation to the subjectivity of human rights in general as well as in relation to specific rights. The interpretation of legal terms "human being" and "everyone" contribute to a better understanding of legal status of embryo in general. The analysis of the specific rights, namely the right to life, human dignity, personal integrity and non-discrimination, shows the complexity of the potential legal subjectivity of embryo in the area of human rights. The evaluation of the legal status of human embryo in the area of human rights protection is complemented by an assessment of legal status in other...
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Criminal aspects of assisted reproduction
Škurlová, Dagmar ; Sovová, Olga (advisor) ; Císařová, Dagmar (referee)
Criminal aspects of assisted reproduction Abstract The work deals with one of the most dynamically developing medical disciplines - assisted reproduction, the introduction and development of their technologies are considered one of the greatest advances in medicine in the 20th century. The purpose of this thesis is to provide the consistent overview of criminal law relations which may arise within providing assisted reproduction and to analyze the individual facts of crimes related to assisted reproduction, which should verify whether their wording provides sufficient protection for relations, interests and values covered by the Criminal Code. The thesis is composed of six chapters. The first chapter presents the fields of criminal law and medical law and examines the relationship between criminal law and health care system. The second chapter lists the conditions of criminal liability of natural and legal persons and lists the ground establishing the absence of criminal liability. The third chapter defines the concept of assisted reproduction, characterizes its methods, provides a historical overview of events that preceded the current interpretation of the concept. The third chapter also provides an overview of the principles that apply in this field of medicine and a list of sources governing the issue....
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The utility of Toll-like receptor 2 in defining the progenitors of definitive embryonic hematopoiesis
Šplíchalová, Iva ; Filipp, Dominik (advisor) ; Nečas, Emanuel (referee) ; Kalina, Tomáš (referee)
Hematopoiesis is a vital process in which red blood cells and cells of the immune system are formed. It is initiated during early embryonic development when we find hematopoietic progenitors in separate anatomical sites. Embryonic hematopoiesis comprises three successive and partly overlapping waves of progenitors with a different hematopoietic potential. The primary anatomical place where hematopoiesis takes place shortly before the birth is the bone marrow (BM). Since at this time point of development BM is already populated by hematopoietic stem cell (HSCs) progenitors, it becomes also the site of hematopoiesis in adulthood. However, the bone marrow is not the only place where hematopoietic progenitors emerge and develop. The Yolk sac (YS) and the Aorta-Gonad-Mesonephros (AGM) region are the initial sites of the appearance of the three waves of progenitors in the early embryogenesis. These progenitors and their descendants play an indispensable role during the development of an individual. Because there are no specific markers that would unambiguously characterize progenitors of these individual waves, their physical separation and hence also functional characterization is still incomplete. Recent studies have shown that Toll-like receptors (TLRs) are expressed on adult HSCs. The stimulation of...
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