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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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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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Isolation and study of proteins with zona pellucida binding activity
Zigo, Michal ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee) ; Antalíková, Jana (referee)
Binding of sperm to the oocyte is mediated by complementary molecules on the surface of both gametes and involves the interaction of sperm protein receptors with the zona pellucida (ZP) saccharide structures. It has been shown that many proteins receptors are involved in the sperm-ZP interaction, and potential primary sperm receptors for ZP glycoproteins have been investigated in various mammals. The majority of proteins with identified sperm-ZP binding activity belong to the plasma membrane proteins. However, the exact methods for isolation of sperm membrane proteins are still to be standardized. This study is focused on investigating how employment of various isolation protocols leads to acquisition of various protein mixtures. Further in the work, two possible approaches towards identification of potential ZP-binding partners are implemented, in order to disclose novel primary ZP-binding receptor candidates. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated by: Triton X-100, Triton X-114, acetic acid, sodium dodecyl sulphate (SDS), N-octyl-β-D- glucopyranoside (OBG), rehydration buffer (RHB), and finally by freezing-thawing extraction and they were characterized by 1-D, 2-D protein profiles, glycoprotein staining and substrate zymographic methods. The results have...
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: Investigation of lymphocyte populations in the porcine small intestine
Kárová, Kristýna ; Šinkora, Marek (advisor) ; Javorková, Eliška (referee)
8 ABSTRACT Historically pig is allocated to a group of animals which use certain parts of their small intestine to acquire a fully developed primary B cell reperoire. Development of such primary repertoire is independent on the antigen presence and resembles the primary lymphopoietic activity of avian bursa of Fabricius. However, some findings concernig the pig's alignment in the above mentioned group suggest otherwise. This graduation thesis is focused on the investigtion of lymphocyte populations and subpopulations in the small intestine of germ-free and conventional piglets. The aim is to determine whether the percentage amounts of lymphocyte populations is dependent on the intestinal colonization. Using Flow Cytometry the significant differences between individual samples were assesed allowing us to conclude which parts of the small intestine could possibly be used for the development of B cell repertoire. Moreover, the status of isotype switching of B lymphocytes isolated from different intestinal parts was determined by the means of PCR analysis. Our data suggest that the small intestine colonization has a crucial role in development of all the main lymphocyte populations as well as some of their subpopulations. The greatest influence of colonization was observed concerning B lymphocytes and their...
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Characterization of the porcine induced pluripotent stem cells
Svobodová, Eliška ; Vodička, Petr (advisor) ; Tlapáková, Tereza (referee)
Induced pluripotent stem cells (iPSCs) are the cells established by introducing several transcription factors into the somatic cells and culturing them in embryonal stem cell (ESCs) culture conditions. Factors used for the establishment of the first iPSCs are OCT3/4, SOX2, KLF4 and c-MYC. IPSCs created by these means resemble closely to the ESCs. IPS technology may be used to derive iPS cells of individual pacients and apply these cells for their treatment in the cases where the use of ESCs represents an ethical and imunological problem. Therefore, it is important to establish an appropriate animal model for the longtime safety testing of iPSCs before acceding to their medicinal aplication.
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Species-specific structural differences of mammalian sperm and function of their key proteins during fertilization.
Dobrodinská, Anna ; Frolíková, Michaela (advisor) ; Kuntová, Barbora (referee)
The fertilization is a process during which a male and a female gamete merge so that a new organism may come into being. The sperm-egg fusion is preceded by several essential processes, such as the capacitation, acrosome reaction, the sperm binding to the zona pellucida and oolemma, and membrane fusion of the gametes. Numerous proteins, which are located in both sperm and eggs, are major actors in controlling the listed, essential processes. During the process of fertilization these proteins fulfil one or more functions. In mammalian sperm, significant species-specific differences may be found both in their morphology and at the protein level. A complex understanding of species-specific distinctions in sperm structure and functions of key sperm proteins would contribute to a better insight into the process of fertilization, thereby enabling us to better diagnose and subsequently treat the causes of infertility in humans. This bachelor's thesis summarizes the current knowledge of sperm structure and its key proteins that has been acquired through the studies of the following model mammal species: bull, boar, mouse, and human. Further, this thesis brings an interspecific comparison between the studied species. Keywords: sperm, fertilization, acrosome reaction, capacitation, sperm proteins, bull,...
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Methods and consequences of some neurosurgical interventions in animal model and in human clinic. Pinealectomy and intracerebroventricular application.
Řezáčová, Lenka ; Tichý, Michal (advisor) ; Szárszoi, Ondrej (referee) ; Přibáň, Vladimír (referee)
Clinical surgical techniques of pinealectomy and intracerebroventricular (i.c.v) cannulation are still used in the clinic in indicated cases. In research, there is an effort to replace these classical surgical techniques by other means. However, these various new models often do not reflect the complexity of the functions taking place in the living organism as a whole. The thesis shows that these surgical techniques should be also a part of biomedical research in the future, as they still bring new important information. In the experiment, the pinealectomy can serve as a model of resection or depletion (removal of natural hormone secretion), while i.c.v. application of the active substance to the ventricles on the contrary as a model of addition or substitution. The practical part of the thesis is divided into 4 areas: A) pinealectomy - an animal models, B) pinealectomy - in the experiment, C) pinealectomy - in the clinical practice and D) intracerebroventricular application - in the experiment. The work describes in detail the surgical techniques and discuss possible consequences of pinealectomy in 6 animal species (and their comparison) and in humans. It also includes an experiment with pinealectomy in a rat and a clinical study in patients with pineal cyst and subsequent pinealectomy. The work...
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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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Opinions of Primary School Students on the Dangers Posed by Selected Mammal Species
Benešová, Šárka ; Andreska, Jan (advisor) ; Hanel, Lubomír (referee)
This thesis builds on my bachelor thesis "Selected mammals in elementary and secondary education". It deals with the opinions of 8th and 9th grade pupils of elementary schools on the dangers posed by selected mammal species. Among the selected species in this work are brown bear (Ursus arktos), wolf (Canis lupus), lynx (Lynx lynx), fox (Vulpes vulpes), dog (Canis lupus familiaris), and wild boar (Sus strofa). All these animals are found in the territory of the Czech Republic. In addition to the perception of dangers posed by these mammals, the thesis also ascertains knowledge of and attitude towards these selected species. The pupils also expressed their opinions about dog breeds and stated whether they currently had a dog, or had had one in the past, and if so, what kind it was. The study was conducted on the basis of a questionnaire survey. Questionnaires were given out at primary schools in areas, where some of the beasts abide (brown bear, wolf, lynx). The work also contains information about individual mammal species that were studied as well as the introduction of the domestic dog, its domestication and an overview of dog breeds. Key words: bear, wolf, dog, lynx, fox, wild boar, breeds
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