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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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Molecular basis of the sperm selection in the mammalian female reproductive tract
Šrámková, Adéla ; Krylov, Vladimír (advisor) ; Postlerová, Pavla (referee)
Internal fertilization is typical for all mammals. The male transfers sperm cells into the female reproductive tract, where usually one of them reach the oocyte and fertilize it. The female reproductive tract must be adapted to provide a suitable environment for the sperm cells to survive until fertilization. On the other hand, it must also be able to select developmentally competent sperm cells and prevent others to get into the close contact with the oocyte. Physical and molecular mutual communication of sperm cells with the female reproductive tract is ensured by sperm surface molecules and the receptors of the female epithelia lining. It appears that these interactions are required for key transformations, both on the sperm surface and in the cells of the female reproductive tract, where gene expression is affected. As a result of these changes, the most suitable sperm cell with all the required characteristics is allowed to fertilize the matured oocyte.
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Mechanisms of the cell migration in the early embryonic development of vertebrates
Kováčiková, Petra ; Krylov, Vladimír (advisor) ; Černý, Robert (referee)
Cell migration, usually seen as a relatively well understood phenomenon, is involved in many aspects of multicellular organisms. While given a major attention due to its part in physiological processes, such as immune response, wound-healing and regeneration, or pathologies like progression of metastatic cancers, its key role in the body plan formation is far less understood and often overlooked. During a process called gastrulation, precursors of three germ layers - superficial ectoderm, inner endoderm and medial mesoderm - are arranged according to their fate, generating a three-layered embryo, a hallmark of triblastic vertebrates. The onset of this highly coordinated step of early embryonic development is accompanied by fine-tuned cell migration, which needs to be downregulated in adults in order to maintain a homeostasis. The cells forming gastrulating embryos collectively undergo a set of behaviors termed morphogenetic movements whose features - partially comparable to ones observed on cells migrating in culture - are shared among vertebrate species, providing valuable insights into "hidden homology" and phylogenetic relationships. Research focusing on regulation and mechanisms of morphogenesis is mostly conducted using Xenopus laevis as a model organism, extrapolating results to higher...
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Functional analysis of invertebrate (Branchiostoma floridae) promoters in heterologous systems
Gurská, Daniela ; Kozmik, Zbyněk (advisor) ; Krylov, Vladimír (referee)
Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...
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Chemical enucleation in mammalian oocytes
Černá, Martina ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The common techniques of enucleation are inexact and time-consuming. Nuclear DNA staining with Hoechst is frequently used, whereas the staining agent is toxic for the oocyte and has negative impact on its further development. Chemical enucleation is a method which facilitates the production of cytoplasts in higher amount and shorter time. Chemical enucleation has a potentional use in the field of biotechnology, especially in nuclear transfer methods (therapeutical, reproductive cloning). The chemical enucleation was successfully used on several model animals: mouse, sheep, cattle. The technique is beeing continually improved on pigs, regarding their similarity to human where it should be consequently applied. It was discovered that same chemicals have different effect on oocytes of each species and therefore it is necessary to examine each species separately. In our study we use porcine oocytes for chemical enucleation. We determined demekolcine in concentration 0,4 Ug/ml and acting time 30 minutes as the most suitable for chemical enucleation. Furthermore we used cytochalasin B in concetration 7,5 Ug/ml and acting time 10 minutes. The protrusion rate correlates with quality of oocytes. In other experiment we focused on distribution of mitochondria after fusion of normal and enucleation oocyte. We found...
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