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LINC complex: The link between chromatin integrity and sperm motility
Šanovec, Ondřej ; Komrsková, Kateřina (advisor) ; Lánská, Eva (referee)
The LINC complex (Linker of the Nucleoskeleton and Cytoskeleton) is a protein structure located in the nuclear membrane that connects the cytoskeleton with the nucleoskeleton. This complex can be found in every mammalian cell including the gametes. However, here the LINC complex is more diverse and less studied than in the somatic cells. In this thesis, the LINC complex and its role in spermiogenesis have been studied in wild-type and Protamine 2 knockout (Prm2-/- ) mice. Protamines are small proteins that replace histones during spermiogenesis. The mouse model generated by the group of prof. Hubert Schorle has a deletion in Prm2 in exon 1 and its sperm possess a surprising phenotype including complete loss of motility. Therefore, it was hypothesized that the LINC complex might be responsible for miscommunication between the sperm head and tail which leads to the loss of sperm motility. Results from this study suggest that the LINC complex is not influenced by Prm2 deletion, however, actin dynamics, cytoskeletal motor proteins and tubulin acetylase/ histone deacetylase activity might be impaired. Prm2-/- sperm have a significantly higher abundance of β-actin compared to the wild type. Next, Prm2-/- sperm also show a different pattern of acetylation of α-tubulin but no change in the abundance of...
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Mapping of the cell population expressing Sonic hedgehog during the embryonic development of heart
Břežná, Veronika ; Hovořáková, Mária (advisor) ; Šaňková, Barbora (referee)
Sonic hedgehog is one of three members of the Hedgehog family, whose signaling pathway plays a crucial role in controlling the development of vertebrates. Sonic hedgehog has an important role in organizing the developmental processes of majority of organ systems. Interestingly, its direct expression or a contribution of cell lineages expressing this signaling molecule in the myocardium has never been described. The aim of this work was to try to detect the cells that expressed Sonic hedgehog directly in the heart tissue with a focus on the myocardium. We monitored these cells from embryonic day 10.5 to 16.5 and then postnatally. We also evaluated current Sonic hedgehog expression in cardiac tissue from embryonic day 12.5 to embryonic day 15.5. We used the CreLoxP system, X-gal staining, fluorescence and confocal microscopy to detect cell lines expressing Sonic hedgehog. We also assessed the presence of cells that expressed Sonic hedgehog in the developping heart in the past using immunohistochemistry. According to our results, the presence of a descendant cell lineage expressing Sonic hedgehog in the past was demonstrated prenatally and postnatally in the mouse myocardium. The performed analysis shows that these cells can be detected in cardiac tissue from embryonic day 10.5 till the postnatal...
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Principles of ovarian stimulation and oocyte collection in mouse and human.
Klinovská, Karolína ; Hortová, Kateřina (advisor) ; Svoboda, Petr (referee)
This thesis deals with methods of controlled ovarian stimulation and oocyte retrieval on physiological basis. Mouse has been major model organism for wide range of key researches and being able to stimulate its ovarian functions correctly is necessary. That is why mouse stimulation is main topic of this thesis. It summarizes available data and stimulation protocols in relation to factors which can affect the result as a condition, strain, quality of hormones used in administration and others. Potential affections on oocyte and embryo quality are mention according to specific types of administration protocols. Human infertility is slowly becoming common and one of the techniques used to treat it in assisted reproduction is also ovarian stimulation. In the end of this thesis is a chapter dealing with this commonly used method. It is focused on stimulation for oocyte retrieval, as well as on stimulation of recipient mother in embryotransfer. Differences based on hormones, success in In vitro fertilization programme (IVF) and application frequency are mentioned.
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Genetic interactions of the Prdm9 gene
Šebestová, Lenka ; Trachtulec, Zdeněk (advisor) ; Král, Jiří (referee)
The Prdm9 gene (PR domain containing 9, Meisetz, Hybrid sterility 1) encodes enzyme that trimethylates histone 3 on lysines 4 and 36. These methylation marks determine the positions of DNA double-strand breaks that are repaired by meiotic homologous recombination. In this study, we assayed genetic interactions of Prdm9 with two genes important for spermatogenesis - Mili (Piwil2) involved in piRNA biogenesis and Mybl1 encoding transcription factor that regulates many genes important for prophase I, including piRNA precursors. We crossed laboratory mice carrying mutation in Prdm9 with heterozygotes for mutation in Mybl1 or Mili, and created compound heterozygotes and, in case of Mybl1, also double homozygotes. We assessed body weight and male fertility parameters (weight of testes, sperm count, malformed sperm, percentage of tubules containing spermatocytes and of abnormal nuclei of pachytene spermatocytes) of these mice and compared them to controls. We also investigated the effect of Mybl1 and Mili mutations on fecundity of F1 intersubspecific hybrids. Our results revealed possible interactions of Prdm9 and Mybl1 in the laboratory mouse. Decreased gene dosage of Mybl1 reduced fertility of intersubspecific F1 hybrids. Interaction between Prdm9 and Mili in both laboratory mouse and F1 hybrids remain...
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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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Morphological variability of the incisor in mutant mice
Lochovská, Kateřina ; Hovořáková, Mária (advisor) ; Churavá, Svatava (referee)
Myš je nejfrekventovaněji používaným experimentálním modelovým organismem pro studium vývoje zubů. Myší funkční dentice obsahuje jeden řezák oddělený od tří molárů dlouhou bezzubou diastemou v každém čelistním kvadrantu. Cílem této práce bylo shrnout poznatky o myší dentici a jejím vývoji se zaměřením na myší řezáky a jejich patologie. Myší řezák je díky svým vlastnostem jedinečným zubem. Charakteristickou vlastností hlodavců jsou právě kontinuálně rostoucí řezáky. Tyto řezáky jsou pokryté sklovinou pouze na labiální straně. Lingvální povrch je tvořen pouze dentinem. Toto je spojeno s asymetrickou abrazí. Nicméně je také častým cílem mutací, ať už v podobě delece nebo jiné modifikace genů. Tyto mutace dentálních signálních drah jsou studovány na mutantních myších jako jsou například Tabby myši, Sprouty nebo Small eye (Sey) mutantní myši a mnoho dalších. Některé mutace jsou homologní k lidským onemocněním. Například X-vázaný tabby (Ta) syndrom u myší je považován za homolog hypohidrotické ektodermální dysplásie (HED) u lidí. Tato mutace napadá velikost řezáku, jeho tvar a pozici stejně jako cytodiferenciaci. Dále se může objevit hypodoncie, anodoncie nebo nějaké morfologické změny napadající ostatní existující zuby. Abnormality v počtu, velikosti a tvaru zubu byly dobře zdokumentovány v lidské...
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Sexual dimorphism in the mouse olfactory system
Kuntová, Barbora ; Stopka, Pavel (advisor) ; Havlíček, Jan (referee) ; Žídek, Lukáš (referee)
Sexually dimorphic behaviour of the house mouse (Mus musculus musculus) relies on various physical and chemical cues, however, chemical signals are the most essential cues for individual recognition and in causing various priming effects on reproductive behaviour of the receiver. House mice belong to macrosmatic mammals, and thus, their sense of smell is highly developed and is able to recognize a wide spectrum of ligands from other individuals and from their surrounding environment. Volatile signals belong to organic compounds that are produced by most tissues, and may have harmfull effects on cells, and thus they are transported out of the body with lipocalin transporters where some of them may function as signals. These volatile signals are able to stimulate chemosensory neuronal receptors, and thus, yield particular responses in neural circuits. The ligand sensing has a differential effect upon males and females, however, it has not been shown yet in wild mice whether these differences are also caused by the variation in receptors and neural processing, or rather by differential expression of signals typical for each sex. The aim of this thesis was to perform comparative analysis of orofacial mucosal tissues to determine the specificity of expression of particular lipocalins. For the first time...
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The signs of odontogenesis externally to the prospective functional dentition in the experimental model
Fábik, Jaroslav ; Hovořáková, Mária (advisor) ; Buchtová, Marcela (referee)
6 Abstract The development of the mouse tooth primordium is an important model for studying odontogenesis, as well as general organogenesis. The development of the mouse lower incisor is of remarkable interest. The epithelial anlage of the mouse lower incisor is interconnected with the vestibular anlage via the epithelial bridges. According to some authors, the epithelial bridges represent an area, where the transient rudimentary incisor germ appears. From a morphological point of view, the first sign of ongoing epithelial-mesenchymal interactions during early odontogenesis is the thickening of oral epithelium. From a molecular point of view, it is the expression of the Shh, Eda, Edar, Pitx2, Bmp2, Bmp4 and Dlx2 genes. Except for the transcription domain, representing the proper signalling centre of a developing prospective incisor, a transient transcription domain, localized anteriorly and superficially, appears in the odontogenic zone of the epithelium during early development. The anterior transcription domain originates in the area of epithelial bridges, and according to some authors, from an evolutionary point of view, it corresponds with the signalling centre of the rudimentary incisor germ. The aim of this diploma thesis was to compare the temporospatial dynamics of SHH and EDA protein expression in...
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Whole-genome analysis of the house mouse hybrid zone
Janoušek, Václav ; Munclinger, Pavel (advisor) ; Choleva, Lukáš (referee) ; Chan, Yingguang Frank (referee)
Hybrid zones provide a valuable opportunity to study the process of speciation in real time. Untested combinations of genes from diverging populations come to the contact here causing a breakdown of genetic interactions and giving rise to reproductive isolation. Two house mouse subspecies (Mus musculus musculus/Mus musculus domesticus) form a narrow zone of secondary contact across Central Europe which is thought to be maintained by a balance between selection against unfit hybrids and dispersion of individuals. During my PhD study my collaborators and I used an array of ~ 1400 SNP markers to study patterns of introgression on a genome-wide scale across two/three house mouse hybrid zone transects. Our aim was to identify the genomic regions putatively harboring genes which are involved in the reproductive isolation between the two subspecies, characterize their distribution in mouse genome and assess genomic features associated with them. We were able to confirm on a genome-wide scale the importance of the X chromosome in the evolution of reproductive isolation. This chromosome exhibited introgression corresponding to strong negative epistasis and the patterns were consistent between transects pointing out to a common basis of reproductive isolation playing a role in two transects. Contrary to the...
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The role of cohesin genes in the meiosis of male house mouse
Šebestová, Lenka ; Trachtulec, Zdeněk (advisor) ; Král, Jiří (referee)
Cohesin genes play an important role in cell division. They ensure proper chromosome segregation during mitosis and meiosis. This study is focused on the role of cohesin genes during meiosis in male house mouse (Mus musculus). At first, this study introduces key processes of mammalian meiosis. Next, the structure of cohesin complex is described; it consists of a heterodimer SMC proteins - SMC3 and SMC1α or SMC1β, which are enclosed to the ring by cleavable subunit RAD21, RAD21L or REC8. Fourth subunit - a STAG protein (STAG1, STAG2 or STAG3) associates with the cleavable subunit. Meiotic function of specific cohesin proteins (SMC1β, RAD21L, REC8 and STAG3) as deduced from the phenotypes of the deficiencies of their genes in male mouse is depicted. All these four genes are necessary for many processes during meiosis, - e.q. sister chromatid cohesion maintenance, synapsis and recombination. STAG3, SMC1β, and REC8 are necessary for centromeric cohesion. STAG3 and RAD21L are important for the assembly of the remaining cohesin subunits. The most important phenotype of deficiency of all four genes is the complete meiotic arrest in male prophase I. Therefore, cohesin research is important for the investigation of the causes of sterility in mammals. key words: cohesin, meiosis, spermatogenesis, mouse,...
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