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Protein synthesis and protein degradation in mammalian oocyte development
Šušor, Andrej ; Motlík, Jan (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Concr,usroNs We anďysed eucaryoticinitiation t,anslation factorsthat are responsiblefor protein qmthesisin mammalianoocýes. As well we examinedthe affinity.of this factorsto.'mCap, Sepharoseduring in útro conďtion...Inour'laboratorywas optimisedthe protocol for generation of poreine parttrenotesthat serve us as a model for anďysis of. cellular processos in .rygotes with biochemical approaches.. : , , - Antibod}r micÍoanay analysis was used to investigate the: regulation of signalling pathways primarily during meiotic maturationof pig oocytesand subsequentlyin comparisonto other species(bovine,frog andseastar). on the base of proteomeanďysis of maturationof oocýes was chosenUCH-LI molebuleandis studiedin detail in mammalian oocyte. Abnost thorougily was analysed function of UCH-LI in porcine and bovine oocyte using specific inhibitors and overexpression. The mechanism that is involved UCH-LI in antipolyspermydefencein mammalian oocyte was unveiled in our laboratory.
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Proteomic architecture of sperm-egg interactions
Otčenášková, Tereza ; Stopka, Pavel (advisor) ; Petr, Jaroslav (referee) ; Vrbacký, Marek (referee)
Recent advances in proteomic methods provide new insights for biological research including the field of reproductive biology. Determination of the proteomic basis of spermatozoa is pivotal for understanding the complex process of gamete interactions during fertilization such as acrosome reaction. Great differences imposed by postcopulatory sexual selection and phylogeny can be observed regarding the size, shape, and molecular composition of sperm across animal taxa. The first objective of this doctoral thesis is to characterize the protein contents of the acrosome to ascertain its further functional significance in sperm-egg interaction. Also, we aim to investigate the potential relationships between sperm protein composition and sperm morphology diversification, risk of sperm competition, and species phylogenetic background. Wild-caught males from natural populations of species of Mus musculus musculus, Apodemus flavicollis, Microtus arvalis (order Rodentia), Acrocephalus palustris, Chloris chloris, Phylloscopus collybita, Cinclus cinclus, Hirundo rustica, and Taeniopygia guttata from a captive population (order Passeriformes) were subject to the analyses. Nano-liquid chromatography with tandem mass spectrometry was applied as the main methodological approach in this thesis. Our data implicate...
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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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Regeneration initiation of Xenopus laevis tail-functional study
Netušil, Jiří ; Šindelka, Radek (advisor) ; Petr, Jaroslav (referee)
The recent introduction of high-throughput sequencing techniques rapidly changes our perception of vertebrate regeneration. One of the model organisms intensely studied for its regenerative potential is the embryonic tadpole stage of African clawed frog (Xenopus laevis). This thesis aims to review and functionally validate some of the latest findings gained by RNA sequencing of regenerating Xenopus laevis tail. We specifically focus on gene expression changes during the early phases of regeneration and how their absence affects the progression and phenotypic outcome of this process. Our analysis confirms that Regeneration initiating cells (RICs) identified by single-cell and spatial RNA sequencing represent a vital element in successful tail regeneration. The RIC marker genes mmp9 and pmepa1 affect the processes linked to extracellular matrix remodelling and the migration pattern of previously described Regeneration organizing cells (ROCs) while not interfering with the myeloid cell lineage. We further demonstrate a novel use of Vivo Morpholino oligonucleotides in a transient knockdown assay and offer an assessment of its benefits and limitations. This thesis highlights the importance of spatio-temporal regulation happening on both cellular and molecular levels during the initial 24 hours of...
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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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Histological Evaluation of Surgical Experiments in Animal Models
Grajciarová, Martina ; Tonar, Zbyněk (advisor) ; Petr, Jaroslav (referee) ; Varga, Ivan (referee)
Introduction: The dissertation is based on six studies that focus on the application of quantitative histology in animal model experiments. It includes a presentation of virtual microscopy procedures and image field sampling strategies, mapping changes in the microscopic structure of ovine and porcine carotid segments and their comparison with human coronary arteries and internal thoracic arteries, vascularization assessment in a mouse model of lymphoma xenografts (PDX), the effect of hyperbaric oxygen therapy on type III collagen production and on vascularization in a skin wound in a Zucker Diabetic Fatty rat. Methods: The review article about virtual microscopy was focused on an example of sampling images from various areas of quantitative histology. In other studies, histologically processed sections were stained with a variety of methods for vascular wall construction, cell infiltration (orcein, picrosirius red, Verhoeff's hematoxylin and green trichrome, Gill's hematoxylin, alcian blue) and immunohistochemical antigen detection (α-smooth muscle actin, neurofilament protein, CD-31, von Willebrand factor). Using unbiased sampling and stereological methods, we quantified the area fraction of components (elastin, collagen, smooth muscle actin and chondroitin sulfate) using a stereological grid...
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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Role of microvesicles and exosomes in reproduction of mammals
Straková, Johana ; Frolíková, Michaela (advisor) ; Petr, Jaroslav (referee)
Microvesicles and exosomes are extracellular vesicles of nanometer size derived from cell membranes. Due to their ability to transfer proteins, lipids and RNA, microvesicles and exosomes are now considered to be a common form of communication between somatic cells. Microvesicles and exosomes have been detected in the reproductive organs of male and female mammals, where they mediate the transport of molecules between cells and thus promote their communication during physiological and pathological processes. Signalling through microvesicles and exosomes is involved in sperm maturation processes, which enrich these vesicles for molecules required for motility and fertilization capacity. Similarly, during oocyte maturation, microvesicles promote communication between follicle cells and its growth. During fertilization, the molecules transported by microvesicles promote capacitation and acrosomal reaction and, after gamete fusion, support the developing embryo and its subsequent implantation in the uterus. The main focus of this bachelor thesis is to discuss the role of microvesicles and exosomes in various processes in mammalian reproduction from sperm and egg maturation to successful fertilization and embryo implantation. A significant part of the thesis is devoted to the presentation of molecules...
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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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Aurora kinase signaling during mammalian oocyte meiosis
Komrsková, Anna ; Drutovič, David (advisor) ; Petr, Jaroslav (referee)
Mammalian oocytes, especially human oocytes, are prone to defective chromosome segregation and are prone to aneuploidies, most commonly in the first meiotic division. Due to these defects, aneuploid embryos are formed after fertilization, resulting in numerous birth defects or infertility. The spindle formation in the first meiotic division in mammalian ooctes is a critical moment for preserving genome integrity. In mammalian oocytes, the spindle assembly is regulated via acentriolar microtubule-organizing centers and chromatine itself, through the activity of Aurora kinases and Ran-GTP gradient. Apart from the Aurora kinase family, other kinases regulate spindle formation, like Polo-like kinase 1 (PLK1). This thesis focused on potential cooperation between Aurora kinases and PLK1 or Ran-GTP. Our results show that the levels of phosphorylated Aurora kinase A (AURKA), but not levels of phosphorylated Aurora kinase B or Aurora kinase C, decrease after pharmacological inhibiton of PLK1. Levels of phosphorylated Aurora kinases do not change after pharmacological inhibition of Ran-GTP signaling. Using live-cell microscopy we showed that overexpression of PLK1 can rescue the phenotype observed in Aurka depleted oocytes. Next, we found that at least one of the signal pathways at chromosomes is necessary...
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