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Regulation of PKB/AKT phosphorylation during resumtion of meiosis
Böhmová, Tereza ; Krylov, Vladimír (referee) ; Šolc, Petr (advisor)
PI3K-PKB signal pathway participates in the CDK1 activation, which is necessary for meiosis resumption of mouse oocytes. That's why we wanted to examine the role of PKB in this process more in the details. The activity of PKB is associated with its phosphorylation at Thr308 and Ser473. These phosphorylations are probably independent and influence PKB function. Thr308 phosphorylated PKB is implicated in resumption of meiosis (GVBD), whereas Ser473 phosporylation is not - as oocytes with reduced phosphorylation on Thr308 have delayed GVBD kinetics and oocytes with inhibited phosphorylation of Ser473 reinitiate meiosis comparably to control oocytes. Conversely, oocyte treatment with synthetic biologically active PtdIns(3,4,5)P3 leads to stronger phosphorylation at Thr308 and accelerated GVBD kinetics. It was also found, that the kinase responsible for Ser473 phosphorylation in mouse oocytes is ATM. Powered by TCPDF (www.tcpdf.org)
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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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Physical mapping of centromeric and terminal markers of linkage groups in Xenopus tropicalis
Tůmová, Lucie ; Krylov, Vladimír (advisor) ; Nedvídek, Josef (referee)
Xenopus tropicalis belongs together with its related genus Xenopus laevis to important model organisms on the field of study of developmental processes. In comparison to well introduced models (i.e. Melanogaster, Mus etc.) for Xenopus tropicalis there is an absence of linkage and physical maps that have invaluable importance for the identification of developmentally relevant genes, including their syntheny with human. Currently, there exists provisional linkage map constructed by scientific group of Department of Biology and Biochemistry, University of Huston, Texas, USA. It is constituted of ten linkage groups (LG1-LG10) and six linkage clusters (A-F) composed by total of 1713 Simple Sequence Repeat (SSR) polymorphic markers. The laboratory of developmental biology, Charles University in Prague, participates in completing the linkage map by physical mapping of chosen polymorphic markers on chromosomes Xenopus tropicalis, which led to allocation of each linkage group and cluster to the right chromosome and to assessment of the orientation in most of them. The physical mapping of terminal markers will show us, how far chromosomes are covered with linkage groups and which areas still remain unmapped. The cooperative laboratory from National Institute for Medical Research, London, UK, originated a list of...
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Study of intronic polymorphisms for the identification of sex chromosomes in Xenopus tropicalis
Seifertová, Eva ; Krylov, Vladimír (advisor) ; Nedvídek, Josef (referee)
10 1.1. ABSTRACT STUDY OF INTRONIC POLYMORPHISMS FOR THE IDENTIFICATION OF SEX CHROMOSOMES IN XENOPUS TROPICALIS Xenopus tropicalis is a significant model organism of developmental biology. Its genom was sequenced. Linkage map based on SSR (Single Sequence Repeat) polymorphic markers was established. The map is still incomplete and contains a lot of inaccuracies. Currently used genetics markers based on repetitive sequences are not spread equally, hence some parts of chromosomes are not genetically mapped. This problem could be solved by polymorphism of intron sequences. To prove its possibly application it is necessary to determine its frequency in genome and confirm its utilization in crossbreeding and consequently in linkage mapping. Beyond its great importance any sex linkage genes are not known in Xenopus tropicalis species, and even a sex type determination is not clear. Gynogenetic studies leading to the determination of sex type produce different accomplishment. Therefore, it is important to create different system enabling sex type determination and, most importantly, to determinate the sex chromosomes. Intronic polymorphisms could serve even this purpose. Keywords: Intron polymorphisms/ Linkage mapping/ Genetic marker/ Sex determination/ Sex gene /Xenopus tropicalis
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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...
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