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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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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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Kryoprezervace a transplantace spermatogonií kapra obecného
FUČÍKOVÁ, Michaela
Cryopreservation and transplantation of germ cells in fish provides a suitable tool for preserving genetic information. By method of surrogate reproduction, the offspring with characters of the chosen donor can be obtained. In this case of our commercially important species common carp. However, for the successful cryopreservation of the germ cells, a suitable protocol for each species must be established. Several cryoprotectants were tested. The best of them, Me2SO, regarding the viability of spermatogonia, was tested for its different concentrations depending also on the rate of freezing. Further testing, related to the effect of tissue size, incubation time and added sugar, was performed. The result of the assay identified best cryomedium composed of 2.5M dimethylsulfoxide, added sugar of 0.3M glucose, 1.5% BSA and 25nM Hepes dissolved in PBS. The most suitable size of tissue was 100 mg, incubation time was 30 min and coolig rate was -1 ° C/min. This protocol ensures the highest viability rate of cryopreserved spermatogonia of common carp. The second part of the work was to verify the success of the transplantation of cryopreserved and fresh spermatogonia into a suitably chosen recipient, the goldfish, which shares similar reproductive characteristics with carp, but also offers reduction of space requirements or resistance to koi-herpes virus. The transplanted germ cells colonized the germ line and started gametogenesis in 42.5% (cryopreserved spermatogonia) and 52.5% (fresh spermatogonia) goldfish recipients, which demonstrated that the transplantation of cryopreserved spermatogonia of common carp can be successfully achieved.
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The rescue of critically endangered fish species through manipulation with spermatogonia and oogonia
DOBROVOLNÝ, Petr
The transplant experiments described in this work may help to shorten the generation interval for long maturing endangered fish species and their more effective reproduction. Further, it is possible to preserve the separated spermatogonia and oogonia using a cryopreservation in liquid nitrogen. We conserve both paternal and maternal DNA and the gene pool of endangered fish species will not be depleted of maternal part. It's because we can freeze only sperm in the preservation of mature gametes. Fish eggs and embryos would not survive freezing. The described methods will be applied in the future to more effective rescue of critically endangered sturgeons by the transplantation of their germ cells into a Sterlet (Acipenser ruthenus). These methods can be suitable for application on other species of endangered fish in case of finding an appropriate recipient. As an example of our fish species is a European eel (Anguilla Anguilla). The Siberian sturgeon (Acipenser baeri) and the Sterlet (Acipenser ruthenus) were used as model organisms. For Siberian sturgeon the enzymatic dissociation technique, sorting of germ cells using Percoll gradient concentration and transplantation of sturgeon spermatogonia and oogonia were used. The results showed that the use of 0.3% of trypsin in PBS is optimal for dissociation of spermatogonia and oogonia, because this medium was dissociating the highest number of cells without reducing their viability. The separation of the early stages of germ cells has been successfully achieved by segregation in 10 to 30% of Percoll gradient with the help of centrifugation. After transplantation it was proved in spermatogonia as well in oogonia that they colonized the genital ridges of the host. The recipient became a chimera of a germ line, which can produce donor gametes throughout his life.
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