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Characterisation of proteins whose degradation is necessary for normal course of embryonic genome activation in mammals
Svobodová, Nika ; Toralová, Tereza (advisor) ; Krejčová, Tereza (referee)
Early embryonic development is controlled by maternal mRNAs and proteins synthesized during oogenesis. A key period for preimplantation development is the transition from maternal control of development to embryonic control. This process, known as maternal-to-zygotic transition (MZT), requires the coordinated degradation of accumulated maternal mRNAs and proteins and subsequent embryonic genome activation (EGA). Maternal mRNAs are gradually removed from the embryo, but the degradation of maternal proteins is not well understood yet. In mammals, only a few proteins are known whose degradation is necessary for normal course of EGA. The activation of the embryonic genome is closely related to the reorganization of chromatin structure. The initiation of gene expression requires the loosening of chromatin at the gene region and the presence of appropriate transcription factors. Maternal proteins that need to be degraded for the normal course of EGA are involved in regulating chromatin structure and the translocation of necessary factors. This thesis aims to characterize selected proteins (PIASy, CBX5, TAB1 and H1FOO) and describe the role they play during the transition from maternal to embryonic control of development. While PIASy, CBX5, and H1FOO need to be degraded to loosen the chromatin structure...
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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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Role paternálního H4K12ac při utváření pronukleí a v časné embryogenezi u myší.
Dudková, Barbora ; Hortová, Kateřina (advisor) ; Stopka, Tomáš (referee)
During the process of spermatogenesis, histones are replaced by protamines, basic proteins enabling transmission of DNA to the oocyte during fertilization. In mouse sperm, there is only 1% of remaining histones whose N-terminal tails contain post-translationally modified residues. In this study, I was interested in contribution of paternal histone H4 acetylated on lysine K12 residues (H4K12ac) that is present in mature sperm head in remaining nucleosomes. Physiologically, H4K12ac has an important role in transcription factor accumulation and in regulation of gene expression. The presence and abundance of H4K12ac modification in various pronuclei stages of 1-cell embryo and parthenotes were assessed by imunnoflourescent detection with utilization of anti-H4K12ac antibody. Altogether, the paternal pronucleus exhibits a strong acetylation signal on H4K12 since its formation, while in the maternal one, there is a slow continual increase of H4K12ac getting on the same level as paternal pronucleus till the pronuclei fusion. Simultaneously DNA methylation status in both pronuclei was detected. In paternal pronucleus there is a continual decrease in the DNA methylation detectable as a decrease of 5mC and an increase of 5hmC signal. Meanwhile, the maternal pronucleus stays widely methylated. DNA...
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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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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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Role paternálního H4K12ac při utváření pronukleí a v časné embryogenezi u myší.
Dudková, Barbora ; Hortová, Kateřina (advisor) ; Stopka, Tomáš (referee)
During the process of spermatogenesis, histones are replaced by protamines, basic proteins enabling transmission of DNA to the oocyte during fertilization. In mouse sperm, there is only 1% of remaining histones whose N-terminal tails contain post-translationally modified residues. In this study, I was interested in contribution of paternal histone H4 acetylated on lysine K12 residues (H4K12ac) that is present in mature sperm head in remaining nucleosomes. Physiologically, H4K12ac has an important role in transcription factor accumulation and in regulation of gene expression. The presence and abundance of H4K12ac modification in various pronuclei stages of 1-cell embryo and parthenotes were assessed by imunnoflourescent detection with utilization of anti-H4K12ac antibody. Altogether, the paternal pronucleus exhibits a strong acetylation signal on H4K12 since its formation, while in the maternal one, there is a slow continual increase of H4K12ac getting on the same level as paternal pronucleus till the pronuclei fusion. Simultaneously DNA methylation status in both pronuclei was detected. In paternal pronucleus there is a continual decrease in the DNA methylation detectable as a decrease of 5mC and an increase of 5hmC signal. Meanwhile, the maternal pronucleus stays widely methylated. DNA...
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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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