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Rab proteins in sperm life
Vojtová, Kristýna ; Páleníková, Veronika (advisor) ; Zelenková, Natálie (referee)
Rab proteins are the largest group of small GTPases. They work as key regulators in the intracellular vesicular transport of cells. This bachelor's thesis contains a literature review of current information on the function of Rab proteins in mammalian spermatozoa. The first part of the work is devoted to their structure, function, cycle, and diseases related to the dysfunction of Rab proteins. The second part is focused on sperm development and maturation linked with specific Rab proteins involved in individual steps during the life of the sperm. Rab proteins are important in cytoskeletal organization and cytokinesis of sperm during spermatogenesis. They participate in the acrosome biogenesis mediated by the Golgi apparatus and the morphological changes of sperm during spermiogenesis. They also play a key role in the acrosome reaction and interaction with the zona pellucida. Due to their necessity in the sperm development and maturation, Rab proteins are proposed as a marker of male fertility, which is summarized in the last part of the thesis. Keywords Rab proteins, GTPases, sperm, spermatogenesis, spermiogenesis, fertilization, infertility
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The role of centrobin in spermatogenesis
Flintová, Jennifer ; Komrsková, Kateřina (advisor) ; Šebková, Nataša (referee)
Spermatogenesis is a highly orchestrated, strictly regulated cascade of events that could be divided into three major processes: mitotic expansion of diploid germ cells (spermatocytogenesis), meiotic division creating haploid cells, and spermiogenesis. Spermiogenesis, the final stage of spermatogenesis comprises a striking metamorphosis of round haploid spermatids into morphologically and functionally specialized spermatozoa designed for the fertilization. One of the proteins indispensable for proper sperm morphogenesis is centrobin, a structural component of the specialized cytoskeletal structures of the elongating spermatids (acroplaxome and manchette), executing essential role in sperm head shaping and assembly of the head-tail coupling apparatus. Disruption in Cntrob gene (coding for centrobin) in rats homozygous at the hd (hypodactyly) locus results in male infertility, with a striking morphological signature called "decapitated sperm syndrome" with detachment of sperm head from the flagellum due to impaired head-tail coupling. However, molecular function of centrobin in spermiogenesis is still unknown. Sperm decapitation is a distinct phenotype described in several mouse mutants and importantly from infertile human males. Strikingly, in addition to proteins functioning in cytoskeletal...
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Role of oxidative stress in male infertility.
Dolečková, Barbora ; Tlapáková, Tereza (advisor) ; Šanovec, Ondřej (referee)
Oxidative stress is a phenomenon caused by an excess of reactive oxygen species (ROS), or by insufficient activity of antioxidants, that reduces these ROS levels and thereby protect the organism from oxidative damage. ROS have two types of origin: endogenous, which includes leukocytes and immature sperm, and exogenous, which includes factors such as air pollution caused by heavy metals, smoking tobacco products, obesity and others. Low levels of ROS have a positive effect on the physiological functions of the organism, including the process of spermatogenesis, where ROS participates in the course of hyperactivation and capacitation. However, increased levels of ROS trigger a number of cellular pathologies, whether the loss of fluidity of biological membranes due to lipid peroxidation, deformation of enzymatic proteins or DNA fragmentation, which negatively affects individuals' infertility. Due to the significant positive correlation of ROS scavenging by antioxidants with improving sperm parameters of an infertile individual, antioxidant therapy has recently begun to be used as a possible successful component of male idiopathic infertility treatment.
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Effect of estrogens on in vitro models of testicular tissue and spermatogenesis
Jursová, Pavlína ; Děd, Lukáš (advisor) ; Tlapáková, Tereza (referee)
Although estrogens are primarily known for their functions in female reproductive system, their effect on male reproductive functions has also been well established. Physiological estrogen concentration is essential for a proper spermatogenesis. Estrogens regulate many functions in testicular tissue, including proliferation and apoptosis of all testicular cell types, dynamic restructuring of cell-cell junctions in the testis, and post-translation modifications of histones. Hence, the aim of this thesis was to study effect of estrogens on in vitro models of testicular tissue and spermatogenesis and thus to address their functions in testicular tissue more deeply. This project includes testicular organoid cultivation for further usage as in vitro model of spermatogenesis. To addresss the effect of various avaliable estrogen forms, experiments on MCF-7 cell line were done. Finally experiments with in vitro model of testicullar tissue - TM4 Sertoli cell line were done. In order to fulfill the aims and verify the hypotheses, some advanced methods such as CLARITY volume confocal imaging and holographic microscopy were used. It was found that estrogens can affect Sertoli cell morphology and the expression of some genes involved in cell-cell junction dynamics. Furthermore the process of spermatogenesis was...
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Sperm maturation process in fishes (review)
KŘIŠŤAN, Jakub
The main aim of this thesis was to review the current knowledge in the field of fish sperm maturation (a process where immature spermatozoa acquire the potential to respond to motility-activating factors) and the urogenital system structure in relation to this maturation.
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Gonadal development during the lifetime of the fastest maturing model vertebrate- turquoise killifish (Nothobrachius furzerí)
LANDOVÁ, Magdaléna
Turquoise killifish had to adapt to the inhospitable conditions in which they live, especially drying temporal water bodies, which means certain death. The life sprint of the representatives of this genus is at its peak within one-month post-hatching, when both sexes have fully developed gonads and can reproduce. This rate comes with a high cost, as the killifish gonads begin to show signs of tissue degradation and germ cell apoptosis as early as three months post-hatching. Germ cell loss increases with age. A description of the development and degradation of the gonads in males and their breeding was elaborated. For the evaluation of aging-specific changes, immunochemical methods were used, focusing on the binding of specific antibodies against target epitopes and their visualization using fluorescence microscopy. Procedures for histological specimens have also been described, both for classical light and fluorescence microscopy.
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Functional Characterization of SCFFBXO38 Ubiquitin Ligase-dependent Protein Degradation
Dibus, Nikol ; Čermák, Lukáš (advisor) ; Konvalinka, Jan (referee) ; D´Angiolella, Vincenzo (referee)
Ubiquitin ligases are responsible for the specific recognition of proteins targeted for proteasome-dependent degradation. This project focused on the molecular and functional characterization of the SCFFBXO38 ubiquitin ligase. As with many others, its biological function has not yet been elucidated in detail, although it is the only ubiquitin ligase whose mutations lead to the onset of a distal form of muscle atrophy. In the first part of our project, we identified new substrates for this ubiquitin ligase, the nuclear proteins ZXDA and ZXDB, with insufficiently characterized functions. Using genetic and biochemical methods, we have shown that ZXDA/B proteins act as positive regulators of centromeric chromatin integrity and that experimental inactivation of the SCFFBXO38 ubiquitin ligase resulted in a ZXDA/B-dependent stabilization of CENP-A and CENP-B proteins in the centromeric regions. In the second part of the project, we focused on analyzing the mouse model deficient in the Fbxo38 gene. We demonstrated that loss of Fbxo38 leads to growth retardation affecting various organs, including the male reproductive system. A detailed histological examination revealed pathological alterations in the seminiferous tubules, accompanied by a lower number of spermatozoa and decreased fertility. We have shown...
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Research of epigenetic aspects of hematopoietic and spermatogenesis stem cells.
Hybešová, Michaela ; Pimková, Kristýna (advisor) ; Děd, Lukáš (referee)
Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...
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Genetic interactions of the Prdm9 gene
Šebestová, Lenka ; Trachtulec, Zdeněk (advisor) ; Král, Jiří (referee)
The Prdm9 gene (PR domain containing 9, Meisetz, Hybrid sterility 1) encodes enzyme that trimethylates histone 3 on lysines 4 and 36. These methylation marks determine the positions of DNA double-strand breaks that are repaired by meiotic homologous recombination. In this study, we assayed genetic interactions of Prdm9 with two genes important for spermatogenesis - Mili (Piwil2) involved in piRNA biogenesis and Mybl1 encoding transcription factor that regulates many genes important for prophase I, including piRNA precursors. We crossed laboratory mice carrying mutation in Prdm9 with heterozygotes for mutation in Mybl1 or Mili, and created compound heterozygotes and, in case of Mybl1, also double homozygotes. We assessed body weight and male fertility parameters (weight of testes, sperm count, malformed sperm, percentage of tubules containing spermatocytes and of abnormal nuclei of pachytene spermatocytes) of these mice and compared them to controls. We also investigated the effect of Mybl1 and Mili mutations on fecundity of F1 intersubspecific hybrids. Our results revealed possible interactions of Prdm9 and Mybl1 in the laboratory mouse. Decreased gene dosage of Mybl1 reduced fertility of intersubspecific F1 hybrids. Interaction between Prdm9 and Mili in both laboratory mouse and F1 hybrids remain...
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Effect of zearalenone on reproductive parameters and the expression of selected genes in mice.
Dvořáková, Eva ; Pěknicová, Jana (advisor) ; Nedvídek, Josef (referee)
A number of chemicals may have a negative impact on the environment and wildlife. Endocrine disruptors (EDs), which can mimic estrogen, interfere with natural hormones in organism and can have a negative effect on the reproductive system. Such substances include zearalenone (ZEA) - mycotoxin, produced by the fungi Fusarium. Despite the non-steriodal structure of its molecule, ZEA and its derivates possess potent estrogenic activity. The influence of ZEA on reproductive parameters and changes in expression of selected genes were tested in the outbred line of mice. This study showed significant effects of ZEA on number of reproductive performances. This effect was observed at lower examined dose, to which are humans normally exposed, and at higher doses, both showed changes in the tested parameters.
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