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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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Chemical enucleation in mammalian oocytes
Černá, Martina ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The common techniques of enucleation are inexact and time-consuming. Nuclear DNA staining with Hoechst is frequently used, whereas the staining agent is toxic for the oocyte and has negative impact on its further development. Chemical enucleation is a method which facilitates the production of cytoplasts in higher amount and shorter time. Chemical enucleation has a potentional use in the field of biotechnology, especially in nuclear transfer methods (therapeutical, reproductive cloning). The chemical enucleation was successfully used on several model animals: mouse, sheep, cattle. The technique is beeing continually improved on pigs, regarding their similarity to human where it should be consequently applied. It was discovered that same chemicals have different effect on oocytes of each species and therefore it is necessary to examine each species separately. In our study we use porcine oocytes for chemical enucleation. We determined demekolcine in concentration 0,4 Ug/ml and acting time 30 minutes as the most suitable for chemical enucleation. Furthermore we used cytochalasin B in concetration 7,5 Ug/ml and acting time 10 minutes. The protrusion rate correlates with quality of oocytes. In other experiment we focused on distribution of mitochondria after fusion of normal and enucleation oocyte. We found...
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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...
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Expressing Causation in the Czech and Spanish Language
Petr, Jaroslav ; Čermák, Petr (advisor) ; Zavadil, Bohumil (referee)
Expressing Causation in Czech and Spanish Language The subject of this thesis, as is evident from the title, consists in expression of causation in Spanish and its equivalents in the Czech language. We dealt with the issue of the causation in both formal and practical perspective. As a basic material, we have used not only Spanish, but also Czech philological publications. We based particularly on the article written by Petr Čermák and Pavel Štichauer called Španělské a italské faktitivní konstrukce hacer/fare + sloveso a jejich české ekvivalenty, as well as from selected chapters of Gramática descriptiva de la lengua española written by different authors. The work is divided into a theoretical part (Chapters 1-4) and a practical part (5-6). In introduction, we set ourselves the goals, we have outlined the research methodology using a parallel corpus InterCorp, which we described in detail. At the theoretical level, we discussed the question of the translation direction and its effect on our research. Furthermore, we defined the terms of causation, causative verb and factitiveness, factitive verb, to be able to continue working with it. We have commented the form of the causative verbs in Czech and their most distinctive features, including a test for distinguishing causative verbs from non-causative verbs....
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