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Charakterizace oocytů afrických rypošů
KOPECKÁ, Eva
This study is focused on characterisation of oocytes of long-lived rodents, naked mole-rat and giant mole-rat, that are emerging models of long-term oocyte and ovarian quality. In the second part, the effect of repeated ovulations on aging-associated quality decline of oocyte is studied, comparing mouse and giant mole-rat.
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Bioinformatic analysis of RNA dynamics during mammalian oocyte-to-embryo transition
Horvat, Filip ; Svoboda, Petr (advisor) ; Bruce, Alexander (referee) ; Vioristo, Sanna (referee)
The oocyte-to-embryo transition (OET) is a complex biological process during which a terminally differentiated oocyte undergoes numerous and coordinated changes to emerge as a collection of totipotent cells - initial blastomeres of a preimplantation embryo. In mammals, this process is primarily controlled through post-transcriptional regulation of maternal RNAs and transcriptional induction of zygotic RNAs. Technological advancements of next-generation sequencing methods during the last decade enabled studying OET through bioinformatic studies of high-throughput transcriptomics and genomics datasets. The work presented in this thesis explores mechanisms of post-transcriptional regulation and dynamics of different RNAs during OET by utilizing in-depth computational analyses. The presented work is divided into three topics, all covering distinct regulatory facets of the mammalian OET and illustrating roles of different RNA species. The first topic discusses dynamics of maternal transcriptomes in mouse. In our research, we explored roles of deadenylase CNOT6L in the maternal mRNA turnover in mice. We provided evidence that animals deficient in Cnot6l expression are subfertile, due to disruption in deadenylation and degradation of maternal mRNAs deposited in the oocyte. In another study, we...
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Ezrin-Radixin-Moesin (ERM) proteins: The link between tetraspanin web and cytoskeleton in gametes
Picková, Jana ; Frolíková, Michaela (advisor) ; Lánská, Eva (referee)
Ezrin, which belongs to the ERM protein family along with radixin and moesin, plays an important role in linking membrane proteins to the actin cytoskeleton. In sperm, ezrin was described so far in bovine and humans, where it probably contributes in actin polymerization during capacitation. During the acrosome reaction, actin plays a significant role in relocation of protein from the inner acrosomal membrane to the equatorial segment, which is an essential process for successful fertilization. One of the proteins that relocates to the equatorial segment, the site of initiation of gamete fusion, is protein CD9. This protein probably stabilizes protein interactions between sperm and oocyte during gamete fusion. In this diploma thesis, we focused on the role of ezrin in linking the CD9 protein to the actin cytoskeleton in mouse sperm. Using indirect immunofluorescence, we detected ezrin, CD9 and actin in acrosome intact mouse sperm. The co-localization of these proteins in the apical acrosomal region suggests their interactions. However, this was not confirmed by our co-immunoprecipitation experiments. Nevertheless, in the apical acrosomal region we also detected EWI-2 protein, which could act as linker between CD9 and ezrin. In this thesis, we also investigated changes in levels of active...
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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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The role of cumulus cells during the maturation of mammalian oocytes
Meniuková, Kateřina ; Krylov, Vladimír (advisor) ; Drutovič, David (referee)
Cumulus cells in the ovarian follicles emerge by differentiation from somatic granulosa cells. They are located around the oocytes in stratified clusters and their innermost layer is called the corona radiata. Cytoplasmic microvilli of cumulus cells pass through zona pellucida to the proximity of the oolema, where formation of gap junctions enables signalling and metabolic codependency. Oocytes produce factors affecting processes in cumulus cells, including their metabolism and viability. By modulating the abundance of cyclic nucleotides in oocytes, cumulus cells allow the maintenance of meiotic arrest, providing time for oocytes to finish their growth and gain competence for maturation. Because oocytes have a very limited ability to process glucose, metabolic cooperation with cumulus cells enables them to gain the energy needed for processes associated with growth and maturation. Several metabolic pathways of glucose processing create energy substrates for the formation of ATP, which they then transport to the oocytes. They also regulate the amount of lipid droplets in oocytes and protect them from cellular toxicity. An increase in the concentration of luteinizing hormone in the follicle just before ovulation induces the transmission of the signal for cumulus expansion. Interruption of the...
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Creation and analysis of conditional knock-out CDK12 for the study of female reproductive ability
Sedmíková, Veronika ; Šušor, Andrej (advisor) ; Frolíková, Michaela (referee)
Oogenesis is a process with a complex interplay of changes at the genetic, cellular, and structural levels that should lead to the differentiation of female gametes. Strict regulation of these processes is required, as any disruption can lead to fertility problems or disabilities in the offspring. The aim of this work is to gain further insight into the processes that influence oocyte development. This work focuses on cyclin-dependent kinase 12 (CDK12), which belongs to the serine/threonine kinase family. It is known for its pleiotropic role in cellular processes such as transcription, translation, cell cycle progression, cell proliferation, DNA damage response and maintenance of genome stability. CDK12 forms an active complex with its binding partner Cyclin K and affects the elongation process of transcription by phosphorylating serine-2 at the C-terminal domain of RNA polymerase II. Previous studies have shown that CDK12 plays a role in blastocyst implantation, deletion of CDK12 in a mouse embryo resulted in embryo lethality, but to my knowledge the function of CDK12 in the oocyte has not been investigated. Our main objective was to create a viable mouse model with conditional knockout of CDK12 using Cre-recombinase expressed under the oocyte specific Zona pellucida-3 promoter to study the...
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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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The role of microRNAs in regulation of mammalian oocyte and embryo development
Marcollová, Kateřina ; Procházka, Radek (advisor) ; Svoboda, Petr (referee)
Cumulus-oocyte complex (COCs) is crucial for mammalian reproduction. Cumulus cells not only nurture the oocyte; they also represent important communicational nodes for mediating information towards and from the oocyte. The non-coding miRNAs can modulate posttranscriptional events they might serve as a useful biomarker for evaluating cell conditions. Based on the COCs staining with vital Lissamine Green B stain we divided cumulus cells into high- and low-quality ones. Furthermore, we implemented division based on the maturation stage, the GV and MII. Sequencing data analysis showed that DE miRNAs from qualitatively different stages donot significantly vary. Nonetheless, significantly DE miRNAs were detected between two developmentally different stages. We identified e.g. ssc-miR-183, ssc-miR-182, and ssc-miR-21-5p to be highly downregulated when comparing GV to MII stage. Among the highly expressed miRNAs from all samples were members of let-7 family (let-7c, let-7a, let- 7f-5p), ssc-miR-16, ssc-miR-21-5p, and ssc-miR-125a. Targeted genes by the DE miRNAs were involved in ErbB, TGF-β, MAPK, FoxO, gap junction and cGMP signalling pathways. We conclude that single miRNAs in cumulus cells probably cannot be used as a reliable oocyte quality marker. On the other hand,changes in the miRNA expression in...
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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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