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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 Impact of Chemotherapy on Male Fertility
Klímová, Natálie ; Frolíková, Michaela (advisor) ; Hynková, Marie (referee)
Abstarct: Chemotherapy, as the primary treatment method for many types of cancer, is associated with a number of side effects, including a potential negative impact on male fertility. The action of chemotherapy interferes with the process of spermatogenesis, a key part of male reproduction, every part of which is highly sensitive to negative influences that can suspend it or permanently damage it. The most sensitive to the effects of chemotherapy are the active mitotic and meiotic phases, during which spermatogenic cells divide and differentiate. These stages are affected because of the high activity of cell division and the increased need for cell growth, a common aspect shared with the cancer cells against which cytostatics are primarily directed. However, chemotherapeutic agents can also affect the function of Sertoli cells and Leydig cells, which are involved in the regulation of spermatogenesis. Infertility observed after chemotherapy treatment can thus be the result of both direct damage to germ cells and indirect damage to endocrine and paracrine control of somatic cells. Thanks to early diagnosis and advances in treatment technologies, the number of successfully cured cancer patients continues to increase. Nevertheless, the protection of male fertility during oncological treatment remains a current...
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Therapeutic influence of Sertoli cells on the effects of oxidative stress in germ cells
Hynková, Marie ; Tlapáková, Tereza (advisor) ; Frolíková, Michaela (referee)
Sertoli cells are somatic cells found in the testes and have a function in supporting spermatogenesis and germ cell nutrition, but also in maintaining an anti-inflammatory environment and forming the blood-testicular barrier in the testes. They are intensively researched for their immunomodulating properties which can be used in transplant medicine or as a therapeutic tool in regenerative medicine. Following previous research confirming that mouse Sertoli cells share some characteristics of mesenchymal stem cells and have multipotent differentiation potential in vitro, experiments were conducted to determine their therapeutic capabilities. Sertoli cells have demonstrated the ability to promote the maintenance of sperm motility during testicular inflammation and reduce the amount of immotile sperm in vivo. Based on preliminary results, they can also reduce the amount of ROS produced by testicular cells affected by oxidative stress in vitro. Keywords: Sertoli cells, germ cells, infertility, oxidative stress, ROS
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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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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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Evolution of sperm morphology in birds
Zemanová, Aneta ; Stopka, Pavel (advisor) ; Frolíková, Michaela (referee)
The sperm is one of the most variable cells that carry various morphological differences across species and is subject to selective pressures that may drive changes in sperm morphology. Different morphological adaptations that emerged may enhance successful fertilization. This thesis focuses on the analysis of sperm morphology in different species of songbirds, the representation of proteins in both sperm and seminal fluid, and their classification into different categories of gene ontology. The main aim of my thesis was to determine whether differences in proteomes follow the level of sperm competition or rather their phylogeny. Six species of songbirds were selected for this study: Marsh Warbler (Acrocephalus palustris), White-throated dipper (Cinclus cinclus), European greenfinch (Chloris chloris), Barn swallow (Hirundo rustica), Common chiffchaff (Phylloscopus collybita) and Zebra finch (Taeniopygia guttata). Sperm morphometric measurements were made in all the species, with the greatest variability recorded in the central part of the sperm flagellum. Using mass spectrometry, were analysed sperm and seminal fluid proteins separately and discriminant analysis was performed to determine the proteins that most differentiated the selected songbird species from each other. Obtained data provide...
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The mechanism of impaired formation of head-tail coupling apparatus in centrobin deficient acephalic spermatozoa
Vlčková, Monika ; Frolíková, Michaela (advisor) ; Krejčová, Tereza (referee)
Acephalic sperm syndrome (ASS) is a rare form of teratozoospermia that is probably genetic in origin. The sperm of individuals with this syndrome have a damaged apparatus connecting the head and flagellum (HTCA), which leads to the formation of acephalic sperm, i.e. sperm with separate heads from flagella. Individuals affected by ASS are almost exclusively infertile. One of the proteins whose mutation causes ASS is SUN5. Together with KASH proteins, SUN proteins are part of the so-called LINC complex, which ensures the connection of the nucleoskeleton with the cytoskeleton. Centrobin is a protein involved in centriole duplication and assembly of the dividing spindle. Rats carrying a defective gene for centrobin have reduced fertility and exhibit an ASS phenotype. For that reason, this model organism was used in this work to study the possible interrelationships of proteins involved in the formation of ASS. Given the similar phenotype of centrobin and SUN5 mutated spermatozoa, there is a possibility that centrobin interacts with one of the LINC complex proteins and together they participate in the formation of the head-flagella junction. Sperm from rats with a defective centrobin gene also show significant damage to the flagella. Decreased Odf1 protein expression has previously been reported in ASS...
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Detection and quantification of maternal RNA localization during early development
Šimková, Kateřina ; Šindelka, Radek (advisor) ; Frolíková, Michaela (referee)
The asymmetric localization of maternal RNAs and proteins is a crucial mechanism for the body plan development in many animal species. These maternal factors are expressed during the oogenesis and they are used for the regulation of early developoment. In this diploma thesis, I addressed the role of asymmetrically localized RNAs along animal- vegetal axis in the early development of Ambystoma mexicanum. The second part of my thesis is focused on RNA localization in dorso-ventral and left-right axes. I identified the localization patterns of many known RNAs along the animal-vegetal axis using RT-qPCR and I also detected several genes, which can be involved in the dorso-ventral or lef-right patterning. Furthermore, we performed transcriptome wide analysis, which revealed changes classified into the following categories: RNAs relocalization, de novo synthesis before the onset of MBT and RNAs degradation during early development. Many vegetally localized genes in Xenopus laevis with the important role in the development of primordial germ cells have been previously described. Surprisingly, my results show that many of these genes are degraded during the early development of A. mexicnum. I believe that this degradation may be due to a different mechanism of PGC development in A. mexicanum and X. laevis....
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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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