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DNA damage response in mammalian oocytes
Vachová, Veronika ; Šolc, Petr (advisor) ; Nevoral, Jan (referee)
During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...
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Effect of triclosan on in vitro meiotic maturation of porcine oocytes
Straková, Johana ; Petr, Jaroslav (advisor) ; Frolíková, Michaela (referee)
Meiotic maturation of oocytes is a key phase in the development of female gametes. The natural physiology of oogenesis and meiotic maturation is currently being influenced by a number of anthropogenic environmental contaminants. These compounds include triclosan. Triclosan (5- chloro-2-(2,4-dichlorophenoxy) phenol) is an antibacterial substance commercially used in cosmetic and disinfectant products. This compound enters the human body by absorption through the skin and mucous membranes. Its presence has been demonstrated in blood, urine, breast milk, liver and adipose tissue. Triclosan is classified as an endocrine disruptor, yet its consumption continues to increase worldwide. The aim of this study is to evaluate the effect of low doses of triclosan on selected markers of meiotic maturation of porcine oocytes in vitro. This work describes the negative effects of triclosan and triclosan in combination with bisphenol S on the meiotic maturation of porcine oocytes in vitro. Furthermore, the effect of these substances on the increased percentage of abnormal meiotic spindles and the stability of the microtubules of the meiotic spindle was demonstrated. Effects on mitochondria and epigenetic effects of triclosan were not proved. The results highlight the risks associated with the commercial use of...
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The effect of Bisphenol S on porcine oocyte meiotic maturation
Kosařová, Zuzana ; Petr, Jaroslav (advisor) ; Kaňka, Jiří (referee)
Meiotické zrání je klíčovou fází vývoje samičích gamet. Do regulace procesu zrání mohou negativně zasahovat látky, které vstupují do organismu z vnějšího prostředí a následně narušují homeostázu na úrovni hormonálního řízení. Tyto látky se označují jako endokrinní disruptory (ED). Mezi nejrozšířenější ED patří bisfenoly. Po prokázání ED aktivity a právních regulacích využití bisfenolu A značně stoupla světová produkce bisfenolu S (BPS). Recentní studie potvrzují negativní vliv BPS přítomného v organismu na reprodukci lidí a zvířat. Přesto zůstává řada dopadů BPS na oogenezi stále nejasná. Tato práce je zaměřena na hodnocení vlivu BPS na vybrané markery rozpadu jaderné membrány v průběhu rozpadu zárodečného váčku (lamin A/C, lamin B1 a LEMD3) a na přeskupování chromatinu po výstupu prasečích oocytů z I. meiotického bloku. Výsledky prokazují negativní vliv BPS na průběh meiotického zrání zrychlením či zpomalením dějů, které souvisejí s rozpadem jaderné membrány a na nutnost se tímto tématem dále zabývat. Klíčová slova endokrinní disruptor, bisfenol S, meiotické zrání, rozpad jaderné membrány, oocyt
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Effect of pergafast 201 on selected markers of in vitro maturation of porcine oocytes.
Procházková, Bára ; Petr, Jaroslav (advisor) ; Benc, Michal (referee)
Pergafast 201 (N-(p-Toluenesulfonyl)-N'-(3-p-toluenesulfonyloxyphenyl)urea) is a patented color developer that is offered as an alternative to the widely used bisphenols A and S (BPA, BPS). These substances have been shown to have a negative effect on human health and reproduction even in very low doses, and they are endocrine disruptors affecting, among other things, the process of meiotic maturation. The effects of pergafast 201 on the mammalian organism during oogenesis have not yet been monitored. The aim of this work was to evaluate the effect of pergafast 201 on selected specific markers of meiotic maturation after exposure of porcine oocytes to pergafast 201 in low concentrations in vitro. This work is the first to describe the negative effects of pergafast 201 on the maturation of porcine oocytes in vitro, confirming the negative effect of pergafast on the course of meiotic maturation and on the ability of the oocyte to reach the final stage of maturation, as well as its effect on the increased incidence of abnormal dividing spindles. The data show the effect of PF201 on the methylation of epigenetic markers H3K4me2 and H3K9me3. Preliminary results also indicate its effect on the mRNA expression of estrogen receptors α and β. Collectively, these results indicate the possible risks...
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Effect of on in vitro maturation of porcine oocytes
Laštůvková, Viktorie ; Petr, Jaroslav (advisor) ; Benc, Michal (referee)
Lysmeral (Lilial, Butylphenyl Methylpropional) is a synthetically produced aldehyde. It is used for its typical lily of the valley-like scent as a perfume ingredient in cosmetic products. It is typically found, for example, in cleaning products. It is on the list of the 26 most well-known synthetic allergens and has been shown to be present in human urine and subsequently in wastewater. According to some opinions, lysmeral should be classified as an endocrine disruptor, which are substances that can interfere with the body's endocrine system. As these substances often affect mammalian reproduction, it is in society's interest to address them, given that they are commonly found in the environment. Lysmeral was not properly tested before being placed on the market and all its effects on organisms are still unknown. The hypothesis of this thesis is that exposure to lysmeral causes undesirable changes in the meiotic maturation of porcine oocytes in vitro and the aim is to investigate the effect of lysmeral, at concentrations corresponding to normal human exposures, on specific markers of meiotic maturation of porcine oocytes. The results confirm the negative effect of lysmeral on the smooth progression of meiotic maturation and the achievement of the final stage of porcine oocyte maturation in vitro....
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The Effect of EGF - like Peptides on Cumulus-Oocyte Complexes
Blaha, Milan ; Němcová, Lucie (advisor) ; Krylov, Vladimír (referee)
In mammals, the ovarian cycle is controlled by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), secreted by the anterior lobe of the pituitary gland. FSH is assumed to be essential for follicle growth. LH is required for the terminal phases of follicle growth, estrogen synthesis, ovulation, development of the corpus luteum and progesterone synthesis. Although cumulus cells and oocytes lack LH-receptors, LH causes cumulus expansion and meiosis resumption. The effect of LH is mediated via EGF-like peptides - amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC). The precursors of these peptides are present in the mural granulosa cells and EGF-like peptides are released by activation of metalloproteases in response to LH-stimulus. The aim of this thesis is to present EGF-like peptides in the wide context of the molecular mechanisms regulating the late phases of folliculogenesis and meiotic maturation of oocytes.
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Role and regulation of nuclear membrane during meiotic maturation of mammalian oocyte
Končická, Markéta ; Kubelka, Michal (advisor) ; Petr, Jaroslav (referee) ; Binarová, Pavla (referee)
Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...
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Bisphenol S influence on selected markers of meiotic maturation of porcine oocytes
Černíková, Terezie ; Petr, Jaroslav (advisor) ; Krylov, Vladimír (referee)
Bisphenol A is a widely used chemical in the manufacture of plastics. The presence of BPA in the environment adversely affects human health due to contamination of air, drinking water and food. Growing concerns about the effects of BPA have led to its regulation in production and development of alternative chemicals to BPA, such as bisphenol S (BPS). However, the effects of BPS were not properly tested before its introduction to production and the effects on human reproduction are still unknown. For this reason, it is desirable to test the effect of BPS on mammalian oocyte development. This study hypothesizes that BPS exposure causes inhibition of meiotic maturation of porcine oocytes in vitro. This study aims to investigate the potency of BPS at low concentrations corresponding to normal human exposures to selected porcine oocyte proteins. The results of this study demonstrate the negative effect of BPS on the progression of meiotic maturation and reaching the mature oocyte stage. In addition, the results show an increase in the formation of defective meiotic spindles and a disruption of mitochondrial integrity after exposure to BPS concentrations. However, the effect of BPS on double-strand breaks was not demonstrated in this study, in contrast to the case of BPA. Taken together, the results show...
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Role and regulation of nuclear membrane during meiotic maturation of mammalian oocyte
Končická, Markéta ; Kubelka, Michal (advisor) ; Petr, Jaroslav (referee) ; Binarová, Pavla (referee)
Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...
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DNA damage response in mammalian oocytes
Vachová, Veronika ; Šolc, Petr (advisor) ; Nevoral, Jan (referee)
During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...
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