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Využití embryotransferu v moderní reprodukci dojeného skotu
Lužíková, Darina
Darina Lužíková, Use of embryo transfer in modern reproduction in dairy cattle The bachelor´s thesis is focused on the use of embryo transfer in moder reproduction in dairy cattle. The main subject of this work is transfer of embryos which are collected in the Netherlands from in vitro production and transfered to the Holstein recipients in the Czech Republic. In the theoretical part is the thesis focused on the methodology of embryo transfer using embryos obtained by the classical way of lavage, including the preparation of donors. This work also describes the most common time and hormonal schedules used in the Czech Republic. The methodology of in vitro embryo production and oocyte retrieval using the ovum pick-up method is also described. The practical part evaluates the results of embryo transfers from the particular farm from December 2018 to April 2021.
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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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Criminal aspects of assisted reproduction
Paták, Milan ; Sovová, Olga (advisor) ; Císařová, Dagmar (referee)
The concept of assisted reproduction is widely discussed by both professional and lay public. New scientific discoveries in the fields of biology and medicine as well as the use of methods and procedures that enable the emergence of new life raise number of issues, not only the legal ones. The purpose of this thesis is to provide the comprehensive survey of criminal law relationships which may arise in the context of providing the assisted reproduction. The thesis examines whether the current legislation guarantees sufficient protection of relations, interests and values covered by the Criminal Code. Within the analysis of crimes the thesis deals with the need for criminal repression and gives proposals de lege ferenda. The thesis is composed of five chapters. The first chapter is subdivided into two parts. Part One defines the concept of assisted reproduction, describes the historical overview of the events that preceded the current interpretation of the concept and explicates basic medical terminology related to the chosen topic. Part Two presents the basic principles on which the Czech regulation is established and contains specification of the relevant sources of law. The second chapter briefly characterizes the criminal and medical law and clarifies their relationship. The third chapter deals with the...
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Gene expression in chicken embryo: micromanipulation and visualization methods
Bendová, Michaela
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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Regeneration initiation of Xenopus laevis tail-functional study
Netušil, Jiří ; Šindelka, Radek (advisor) ; Petr, Jaroslav (referee)
The recent introduction of high-throughput sequencing techniques rapidly changes our perception of vertebrate regeneration. One of the model organisms intensely studied for its regenerative potential is the embryonic tadpole stage of African clawed frog (Xenopus laevis). This thesis aims to review and functionally validate some of the latest findings gained by RNA sequencing of regenerating Xenopus laevis tail. We specifically focus on gene expression changes during the early phases of regeneration and how their absence affects the progression and phenotypic outcome of this process. Our analysis confirms that Regeneration initiating cells (RICs) identified by single-cell and spatial RNA sequencing represent a vital element in successful tail regeneration. The RIC marker genes mmp9 and pmepa1 affect the processes linked to extracellular matrix remodelling and the migration pattern of previously described Regeneration organizing cells (ROCs) while not interfering with the myeloid cell lineage. We further demonstrate a novel use of Vivo Morpholino oligonucleotides in a transient knockdown assay and offer an assessment of its benefits and limitations. This thesis highlights the importance of spatio-temporal regulation happening on both cellular and molecular levels during the initial 24 hours of...
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Rypoš lysý a myš domácí jako modelové organismy reprodukčního stárnutí
KOPECKÁ, Eva
The aim of this study is to demonstrate the naked mole rat as a new possible model of reproductive aging. It is also pointing out the reproductive problems and diseases associated with increasing age in women. Furthermore, the study wants to introduce the role of mTOR in mouse embryonic development in relation to maternal age and decrease in fertility in a view of number of oocytes in mouse, guinea pig and naked mole rat.
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The foundation of maternal factors in sturgeon: from oocyte to embryo
POCHERNIAIEVA, Kseniia Kostyantynivna
The effective application of embryo engineering to endangered sturgeon species requires fundamental knowledge of its embryonic development and information about structure and characteristics of sturgeon oocyte itself. To reveal intracellular geometry, mechanisms of maternal determinants organization and its later reorganization and morphogenetic aspects we used several techniques such as qPCR tomography, inhibition of transcription and visualization of nucleuos. The qPCR tomography was discovered as reliable technique to determine the role of the genes detected in the animal and vegetal hemispheres of the sturgeon oocyte, and to identify profiles of these genes during early developmental stages of sturgeon embryos. The 12 selected maternal genes were investigated. Two groups of transcriptomes categorized as animal or vegetal with evident gradient profile were identified. The primarily germplasm markers such as dnd, vasa, ddx25 were localized toward the extreme vegetal pole. This finding reveals localization of primordial germ cells in the body plan of the sturgeon oocyte. Another aspect of applying such technique was comparative analysis of RNA profiles in the oocyte of distantly-related species Xenopus laevis and Acipenser ruthenus. We found clear similarity in the localization of mRNA molecules in Acipenser ruthenus and Xenopus laevis, which revealed significant aspects of early development that have been conserved during evolution. Such similarities in expression profiles of distantly related species indicate that their ancestors could have arisen from more closely related lineages. The maternal to zygotic transition (MZT) is a separate developmental period that begins with the elimination of maternal transcripts, continues through the production of zygotic transcripts, and concludes with the first major morphological requirement for zygotic transcripts in embryo development.The alpha-Amanitin as transcript inhibition factor was used to determine the zygotic genome switch in sterlet embryos. The transition in sterlet was observed after the tenth cleavage during late blastula, when blastomeres in the animal pole are surpassed 1000 cells. Mid-blastula transition (MBT) in early embryogenesis can be defined as a time point characterized by cell cycle lengthening, loss of synchrony and acquisition of cell motility. We opted to use oocytes of crosses sterlet A. ruthenus and Russian sturgeon Acipenser gueldenstaedtii, since the hybridization results in increased DNA content in their hybrid offspring compared to parental species A. ruthenus making the embryo a useful model for investigation of changes in the timing of early development. Nucleous vizualization by 4'-6-diaminido-2-phenylindole (DAPI) staining showed that cells divided synchronously at a constant rate until MBT at the ninth cell cycle in control sterlet embryos that corresponds to 1000 cell stage (13 hpf). The sterlet x Russian sturgeon hybrid embryos showed transition from synchronous to asynchronous division at the eighth cell cycle which is the 512 cells stage (12 hpf). In both sterlet and hybrid embryos, the transition occurred within 1 h. Thus, our study confirmed hypothesis the MBT in sturgeon is governed by the ratio of nucleus to cytoplasm, which can be controlled using hybridization, induction of polyspermy or injecting plasmid DNA Embryos of sturgeon injected with alpha-Amanitin also showed cell cycle kinetics similar to controls, with no delay or malformation during cleavage, which most likely indicates that MBT in the sturgeon proceeds independently of onset of zygotic transcripts production. The results and observations presented in this study demonstrate the path from an egg to a developed embryo, which are the basis for improving the production methods and preservation of sturgeons listed in the IUCN Red List, and which is equally important, provide the fundamental knowledge about the nature of sturgeons.
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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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