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Endogenně produkovaný sulfan v reprodukčním traktu samice ve vztahu k fertilizaci
BRICHCÍN, Jiří
The discovery of the mechanisms responsible for the proper process of fertilization and early embryonic development helps in the development of assisted human reproduction and technologies in livestock breeding. One of the regulatory mechanisms involved in reproduction is the gasotransmitter hydrogen sulfide, whose cellular signaling in germ cells is not fully described yet. According to the hypothesis, hydrogen sulfide is released in the female reproductive tract, where it physiologically affects sperm and is necessary for fertilization and the development of pronuclei. The aim was to verify the expression of hydrogen sulfide-releasing enzymes and the existence of a time-space gradient of hydrogen sulfide within the estrous cycle of the mouse (time axis) and to compare the expression profile of the fallopian tube ampulla and uterotubal junction (space axis). The experiments were performed on a laboratory mouse model (Mus musculus). The expression of hydrogen sulfide-releasing enzymes was studied by western blot. Hydrogen sulfide production was determined by colorimetry. The role of hydrogen sulfide in the fertilization process was studied by in vitro fertilization with hydrogen sulfide donor-treated sperm and by immunocytochemical staining of the zygotes. The expression of the known hydrogen sulfide-releasing enzymes, i.e. cystathionine--lyase (CTH), cystathionine-ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MPST), was detected within the ovary and fallopian tube. Any enzymes did not show statistically significant differences in estrus and diestrus expression. Moreover, analogous results were found in the spatial axis; both the fallopian tube and the uterotubal junction expressed CTH, CBS, and 3-MPST enzymes, but no statistical differences were noted. Also, this applies to the production of hydrogen sulfide by these tissues. The effect of hydrogen sulfide on the fertilization process was analyzed according to lamin B1, a paternal pronucleus development marker. The performed analysis did not confirm the influence of the hydrogen sulfide donor on the development of the zygote pronuclei. The work showed that hydrogen sulfide is enzymatically released by the tissues of the female reproductive tract, regardless of the phase of the estrous cycle or the location in the fallopian tube. There is assumed a physiological influence of maternal-born hydrogen sulfide to sperm, which is capable of fertilization and leads to pronuclei development.
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Gasotransmise v epigenetických regulacích gametogeneze a embryogeneze
BRICHCÍN, Jiří
As the problems with the reproduction of livestock and humans starting to increase, the need for knowledge of mechanisms involved in regulating the correct process of gametogenesis and embryogenesis also rises. For the experimental part of this work, two components from two different, mostly separately explored fields, i.e. gasotransmitters and epigenetic mechanisms, which are necessary for the correct process of gametes production and early embryonic evolution were chosen. Hydrogen sulfide was chosen from the series of gasotransmitters, and histone deacetylase Sirtuin 1 (SIRT1) was chosen as its possible substrate. Confirmation of the presence of these components was carried out on oocytes and embryos of laboratory mice (Mus musculus).
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Quantum-chemical study of noncovalent interactions
Sedlák, Róbert ; Hobza, Pavel (advisor) ; Havlas, Zdeněk (referee) ; Černušák, Ivan (referee)
The aim of this thesis is to investigate strength and origin of the stabilization for various types of noncovalent interactions. As this knowledge could lead to a deeper understand- ing and rationalization of the binding phenomena. Further, to participate on the de- velopment of new noncovalent data sets, which are nowadays inevitable in the process of parametrization and validation of new computational methods. In all the studies, different binding motifs of model complexes, which represent usually crystal structures, structures from unrelaxed scans or the local minima, were investi- gated. The calculations of the reference stabilization energies were carried out at ab initio level (e.g. CCSD(T)/CBS, QCISD(T)/CBS). Further, the accuracy of more ap- proximate methods (e.g. MP2.5, DFT-D or SQM methods) toward reference method, was tested. In order to obtain the nature of the stabilization the DFT-SAPT decompo- sition was frequently utilized. In the first part of the thesis, the importance and basic characteristics of different types of noncovalent interactions (e.g. halogen bond, hydrogen bond, π· · · π interaction etc.), are discussed. The second part provides the description of computational methods which were essential for our investigation. The third part of the thesis provides an overview for part...
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Intracellular distribution of hydrogen sulfide releasing enzymes in maturing porcine oocytes
Vondráková, Veronika ; Hošková, Kristýna (advisor) ; Tomáš, Tomáš (referee)
The study of the molecular mechanisms regulating reproductive processes is crucial for increasing the efficiency of reproductive biotechnologies that are often used in animal breeding. A key part of many biotechnological methods is the cultivation of oocytes, which simulates natural conditions in the ovaries. However this simulation is still imperfect and presents a limiting factor for successful formation of mammalian oocytes.capable of fertilization.
Meiotic maturation is influenced by many factors including for example gasotransmiter hydrogen sulfide (H2S). Until recently, hydrogen sulfide was known only as toxic gas polluting the environment. It was found that the hydrogen sulfide is at low concentrations important signaling molecule with a lot of physiological functions. Hydrogen sulfide is produced by enzymes CBS, CTH and 3-MPST in various tissues of the body, including the reproductive system.
We hypothesised that enzymes responsible for the endogenous production of hydrogen sulfide, CBS, CTH and 3-MPST, are present in porcine oocytes and their presence varies during meiotic maturation in cellular organelles. The goal of this study was to prove this hypothesis by cultivating porcine oocytes, detection of mitochondria, endoplasmic reticulum and hydrogen sulfide-releasing enzymes using imunocytochemistry and colocalization analysis.
Our results showed that enzymes releasing hydrogen sulfide, CBS, CTH and 3-MPST, are present in GV porcine oocytes and also in the oocytes matured to the MII stage, after 48 hours in vitro cultivation. We found that the distribution of these enzymes in cellular organelles in oocyte changed during meiotic maturation. There were significant changes in the distribution of enzymes 3-MPST and CBS in the mitochondria, levels of colocalization coefficients for enzyme 3-MPST increased, these coefficients for enzyme CBS decreased during meiotic maturation. In endoplasmic reticulum we found significant changes in the distribution of all three hydrogen sulfide-releasing enzymes. Levels of colocalitazion coefficients for CBS and 3-MPST increased, while levels of these coefficients for enzyme CTH decreased. Results of our experiments on porcine oocytes can help to clarify the role of gasotransmitter hydrogen sulfide during meiotic maturaion mammals of oocytes and are highly valueable because their possible application in breeding of farm animals and in human assisted reproduction.
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Interactions of Hydrogen Sulfide Releasing Enzymes in Porcine Oocytes during Meiotic Maturation
Jiříček Hasalová, Simona ; Hošková, Kristýna (advisor) ; Tomáš, Tomáš (referee)
The reproductive biotechnologies currently represent a major scientific discipline based on a sufficient quantity of wholesome oocytes matured in vitro conditions to acquire permits for their effectiveness. The meiotic maturation is a complex process where a wide range of factors is involved in the regulation. One of these factors are small gaseous molecules, so-called gasotransmitters. These gases demonstrate number of physiological functions in the organism and the latest discovered gasotransmitter is hydrogen sulfide (H2S).
The aim of this thesis was to test the hypothesis according to which there are enzymes present in porcine oocytes that produce H2S (CBS, CTH, 3-MPST), their distribution is in interaction and their joint actions influence the process of the meiotic maturation. The resulting data were obtained on the basis of immunocytochemical staining and colocalization analysis.
The results proofed the presence of H2S-releasing enzymes in porcine oocytes. It was also confirmed the function of these enzymes in relation to the regulation of the meiotic maturation when oocytes cultured with triple inhibitors of H2S-releasing enzymes matured more slowly. The results also showed medium correlation dependence of enzymes CBS, CTH and 3-MPST. The combination of CBS and 3-MPST resulted in high correlation dependence which confirmed their interaction both in immature oocytes and in vitro matured oocytes.
It is evident that so far studied effects of H2S are only a fraction of skills which this signal molecule possesses. There exists a need for future experiments to help us describe and explain the acting mechanisms of H2S.
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