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In vitro kultivace zárodečných kmenových buněk jesetera
XIE, Xuan
The study in this thesis concerns a research area related to sturgeon germ cell biology including identification, purification and in vitro culture. The research objectives are: expounding fish spermatogonial stem cell characteristic and regulation of spermatogenesis; evaluation of the existent methods in fish germ cell purification and in vitro culture; investigation of in vitro amplification of sturgeon germ cells; development of an approach to purify germ cells in sturgeon; identification of type A spermatogonia (ASG) in sturgeon. Novelty and scientific originality. This work first time developed sturgeon germ cell growth in vitro and combined cell culture with transplantation. The application of monoclonal antibodies on recognition of sturgeon ASG represents a new, and currently only tool of germ cell identification. Important scientific problem solved in the field of research. This work of germ cell culture observed the effect of somatic cells and growth factors on the proliferation of sturgeon germ cells through evaluation of germ cell mitotic activity during culture, which contributed to the study on the regulation mechanism of germ cell self-renewal and differentiation. This thesis also developed a method using flow cytometry to purify sturgeon ASGs. A monoclonal antibody was also found that enable to identify ASGs of sturgeon specifically. Theoretical significance and applied value of the thesis. The thesis summarized the accumulated knowledge of spermatogonial stem cell and regulation of its self-renewal vs. differentiation in fish, compared available protocols and advances in enriching spermatogonia and reviewed development of in vitro germ cell culture conditions. The work of efficient enrichment of type A spermatogonia was achieved based on its physicochemical properties, which is applicable for commercial and endangered fishes without cell-labeling systems such as transgenes and cell antibodies. Monoclonal antibodies generated from Pacific blue fin tuna can also recognize antigens from sterlet ASGs, indicating some epitopes for ASGs are conserved between species. The research results present interest for characterization of ASGs in proteomics level. Implementation of scientific results. A serum-free culture condition for sturgeon germ cells was established. The germ cells under this in vitro condition can remain growth, survival and maintain their transplantability for more than 40 days. Therefore, this method is expected to provide a long term supply for germ cell transplantation and surrogate production. Moreover, based on the analysis of germ cell light scatter properties, a purified ASG population was isolated. No dye and transgenes are introduced during sorting, which is helpful for characterization of germ cell populations and downstream germ cell manipulation. The application of monoclonal antibody on sturgeon ASG can achieve the accuracy of the ASGs identification on in vitro culture.
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The foundation of maternal factors in sturgeon: from oocyte to embryo
POCHERNIAIEVA, Kseniia Kostyantynivna
The effective application of embryo engineering to endangered sturgeon species requires fundamental knowledge of its embryonic development and information about structure and characteristics of sturgeon oocyte itself. To reveal intracellular geometry, mechanisms of maternal determinants organization and its later reorganization and morphogenetic aspects we used several techniques such as qPCR tomography, inhibition of transcription and visualization of nucleuos. The qPCR tomography was discovered as reliable technique to determine the role of the genes detected in the animal and vegetal hemispheres of the sturgeon oocyte, and to identify profiles of these genes during early developmental stages of sturgeon embryos. The 12 selected maternal genes were investigated. Two groups of transcriptomes categorized as animal or vegetal with evident gradient profile were identified. The primarily germplasm markers such as dnd, vasa, ddx25 were localized toward the extreme vegetal pole. This finding reveals localization of primordial germ cells in the body plan of the sturgeon oocyte. Another aspect of applying such technique was comparative analysis of RNA profiles in the oocyte of distantly-related species Xenopus laevis and Acipenser ruthenus. We found clear similarity in the localization of mRNA molecules in Acipenser ruthenus and Xenopus laevis, which revealed significant aspects of early development that have been conserved during evolution. Such similarities in expression profiles of distantly related species indicate that their ancestors could have arisen from more closely related lineages. The maternal to zygotic transition (MZT) is a separate developmental period that begins with the elimination of maternal transcripts, continues through the production of zygotic transcripts, and concludes with the first major morphological requirement for zygotic transcripts in embryo development.The alpha-Amanitin as transcript inhibition factor was used to determine the zygotic genome switch in sterlet embryos. The transition in sterlet was observed after the tenth cleavage during late blastula, when blastomeres in the animal pole are surpassed 1000 cells. Mid-blastula transition (MBT) in early embryogenesis can be defined as a time point characterized by cell cycle lengthening, loss of synchrony and acquisition of cell motility. We opted to use oocytes of crosses sterlet A. ruthenus and Russian sturgeon Acipenser gueldenstaedtii, since the hybridization results in increased DNA content in their hybrid offspring compared to parental species A. ruthenus making the embryo a useful model for investigation of changes in the timing of early development. Nucleous vizualization by 4'-6-diaminido-2-phenylindole (DAPI) staining showed that cells divided synchronously at a constant rate until MBT at the ninth cell cycle in control sterlet embryos that corresponds to 1000 cell stage (13 hpf). The sterlet x Russian sturgeon hybrid embryos showed transition from synchronous to asynchronous division at the eighth cell cycle which is the 512 cells stage (12 hpf). In both sterlet and hybrid embryos, the transition occurred within 1 h. Thus, our study confirmed hypothesis the MBT in sturgeon is governed by the ratio of nucleus to cytoplasm, which can be controlled using hybridization, induction of polyspermy or injecting plasmid DNA Embryos of sturgeon injected with alpha-Amanitin also showed cell cycle kinetics similar to controls, with no delay or malformation during cleavage, which most likely indicates that MBT in the sturgeon proceeds independently of onset of zygotic transcripts production. The results and observations presented in this study demonstrate the path from an egg to a developed embryo, which are the basis for improving the production methods and preservation of sturgeons listed in the IUCN Red List, and which is equally important, provide the fundamental knowledge about the nature of sturgeons.
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Differentiation of totipotent germinal cells in larvae of bird schistosomes
Peštová, Jitka ; Horák, Petr (advisor) ; Chanová, Marta (referee)
This thesis aims to explore the larval development of a bird fluke Trichobilharzia regenti in its intermediate hosts, as well as the processes of differentiation of its embryonal cells and the differentiation between sporocystogenesis and cercariogenesis in sporocysts, with the ultimate goal to find out whether it is possible to find multiple generations of daughter sporocysts throughout the development of avian schistosomes in the intermediate hosts, just like in the case of human schistosomes of genus Schistosoma. Five developmental stages of daughter sporocysts, and ten developmental stages of cercariae have been defined. The first developmental stage in both larvae is the germinal cell. It divides and gives rise to a cell agregate. Afterwards an envelope (primitive epithelium) is formed around the embryo and subsequently, the embryo elongates. At this stage, the development of the two larvae undergoes different pathways. We can distinguish daughter sporocyst from cercaria in the phase, when the tegument is completed. The daughter sporocyst acquires characteristic vermiform appearance, and its body cavity contains plenty of germinal cells. For cercariae with an developed tegument, presence of the penetration glands is characteristic. Key words: Trichobilharzia regenti, germinal cells, mother...
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Manipulace zárodečných buněk jako nástroj pro management a produkci izogenních linií u ryb
FRANĚK, Roman
Isogenic lines in fish represent a fundamental approach to control the genetic background of experimental animals. All individuals from a given isogenic line share the same genotype. So far, isogenic fish lines have been produced only by repeated uniparental inheritance - androgenesis and gynogenesis. Homozygous progeny is produced in the first generation of uniparental inheritance, and each homozygous individual produces a different isogenic line after second generation of uniparental inheritance. Despite optimized procedures for inducing uniparental inheritance, isogenic lines have been successfully produced in only a few species of fish. Doubled haploids after first uniparental inheritance have affected fitness as well as reproductive performance. Long-term maintenance is considerably problematic even when isogenic line is established already, due to low viability and poor reproductive characteristics. The situation is further complicated by the fact that isogenic lines are usually naturally monosex, thus uniparental inheritance must be re-used for further reproduction, or sex reversal needs to be applied in part of isogenic line. Several types of germ cell manipulation were performed in presented thesis. Protocols for cryopreservation of spermatogonia and oogonia have been developed and optimized to maximize post-thaw viability. The physiological activity of cryopreserved cells was confirmed by transplantation into a surrogate host. Cryopreserved and subsequently transplanted cells retained colonization activity comparable to non-frozen control germ cells. More importantly, male germ cells were able to transdifferentiate from oogonia. The success of transplantation was confirmed by detection of expression of genes associated with gametogenesis in carp by RT-PCR. In the next study, the results of cryopreservation experiments were followed, where sterile goldfish was identified as a suitable host for homozygous carp cells. Germ cells obtained from several homozygous individuals were individually transplanted into sterile goldfish. This procedure has a potential to increase the chance of producing a viable gamete for isogenic line production. Germ cells from homozygotes with affected gametogenesis can be transferred to fully viable recipients, thereby increasing the efficiency of isogenic line production overall. In addition, the use of a goldfish as a surrogate parent will ensure that part of the germline chimeras will be male and female, thus isogenic gametes of both sexes can be obtained and no further intervention for further reproduction of the isogenic line. The suitability of triploid zebrafish, which can potentially be used as recipients for cells from homozygotes to produce isogenic lines, has been confirmed for zebrafish. Spermatogonia and oogonia from diploid donors were transplanted into artificially induced triploid larvae. Donor-derived sperm was were obtained upon maturation of triploid recipients. Transplanted oogonia transdifferentiated into spermatogonia and spermatozoa with female sex chromosomes have been produced, which may be of interesting for further studies of sex determination in zebrafish. A new germline transfer technique has been developed using striped embryos. Donor cells were transplanted from the blastula stage to the swim-up larvae. With this approach, undifferentiated primordial germ cells were able to colonize the genital groove and initiate gametogenesis. After reaching sexual maturity, germline chimeras were obtained with gametes and viable progeny. Although the overall efficacy of this method was lower compared to other transplantation methods, this study may be of relevance for germline rescue in poorly viable embryos or lethal mutants.
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Legal issues of gametes in relation to assisted reproduction
Stieranková, Aneta ; Frinta, Ondřej (advisor) ; Thöndel, Alexandr (referee)
66 Abstract Legal issues of gametes in relation to assisted reproduction The thesis deals with the issue of assisted reproduction with a specific focus on gametes and some issues that are associated with them. In order to better understand contradictory opinions and views on a particular issue, the Czech legislation is compared with the British legislation. Then, in each individual case, it is assessed which country has dealt with the problem better, using a comparative method. The introduction of the thesis deals with assisted reproduction in general, especially from the point of view of definition of the concepts and historical development of this issue. Subsequently, the basic legal framework for assisted reproduction is defined both in the Czech Republic and the United Kingdom. Briefly, there are also summarized the most basic requirements for gametes donors in both countries. In terms of specific problems related to gametes, much of the work focuses on the anonymity of sperm donors, in particular summarizing the most important arguments of its opponents and subsequently refuting these arguments. The conclusion of this chapter submits why, in my view, the anonymity of sperm donors should be maintained. The next part is devoted to financial rewards for donation of gametes, their admissibility and amount....
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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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Differentiation of totipotent germinal cells in larvae of bird schistosomes
Peštová, Jitka ; Horák, Petr (advisor) ; Chanová, Marta (referee)
This thesis aims to explore the larval development of a bird fluke Trichobilharzia regenti in its intermediate hosts, as well as the processes of differentiation of its embryonal cells and the differentiation between sporocystogenesis and cercariogenesis in sporocysts, with the ultimate goal to find out whether it is possible to find multiple generations of daughter sporocysts throughout the development of avian schistosomes in the intermediate hosts, just like in the case of human schistosomes of genus Schistosoma. Five developmental stages of daughter sporocysts, and ten developmental stages of cercariae have been defined. The first developmental stage in both larvae is the germinal cell. It divides and gives rise to a cell agregate. Afterwards an envelope (primitive epithelium) is formed around the embryo and subsequently, the embryo elongates. At this stage, the development of the two larvae undergoes different pathways. We can distinguish daughter sporocyst from cercaria in the phase, when the tegument is completed. The daughter sporocyst acquires characteristic vermiform appearance, and its body cavity contains plenty of germinal cells. For cercariae with an developed tegument, presence of the penetration glands is characteristic. Key words: Trichobilharzia regenti, germinal cells, mother...
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Mikromanipulace a kryopreservace zárodečných buněk ryb
LINHARTOVÁ, Zuzana
The induction of germ-line chimerism is an expanding focus of fisheries research. This technique is having a potential to enhance the production of gametes of species that are commercially valuable, endangered, species with problematic reproduction, using a more common or easily available species or species adapted to artificial reproduction as a surrogate host. The main goal of this technology is to establish a small-bodied surrogate broodstock producing functional donor gametes based on germ cell transplantation. Extent preliminary experiments, including documentation of donor/host embryonic and larval development, characterization of germ cells enriched by documentation of their migratory activities, sterilization of the host, isolation and cryopreservation of donor germ cells, are key factors for launching this biotechnology. All these crucial points were the main objective of the present work. The whole thesis provided the focus on two different fish species. First, our commercially valuable fish, the tench, where we would like to apply our current knowledge to create a germ-line chimera within cyprinids by transplantation of tench germ cells to smaller and faster-reproducing fish species as white cloud mountain minnow. Secondly we focused on the endangered species (listed in IUCN Red List) of large body size with long reproductive cycle, the sturgeons. In this case, we have chosen sterlet as a host, providing an advantage of shorter generation interval and smaller body size, to produce gametes of donor, a critically endangered species of large body size with long reproductive cycle, such as beluga. This innovative technology could result in collection of sperm and eggs in shorter time from small-bodied host. In tench we firstly focused on embryonic and larval development documentation together with description of origin and migration pathways of primordial germ cells (PGCs). PGCs represent a powerful tool for creation a germ-line chimera within fish species because they transmit genetic information to the next generation (Linhartova et al., 2014a). Secondly, we reported a practical technique for isolation and cryopreservation of early stages of germ cells (GC), including spermatogonia (SG) and spermatocytes (Linhartova et al., 2014b). In case of sturgeons, Saito et al. (2014) firstly described the origin and migration patterns of sturgeon PGCs deposited at the vegetal pole of the egg similar to that in anurans. Secondly, Psenicka et al. (2015) reported isolation and cryopreservation of female and male GC, SG from testes and OG and pre-vitellogenic oocytes from ovary, of 2-4-year old Siberian sturgeon. Moreover the isolated GC were transplanted into host (sterlet) and process of transplantation resulted in successful colonization of sterlet genital ridge. The potential host for germ-cell tranplantation, sterlet, was sterilized by knock-down of germ cell specific gene, the dead end gene, by the morpholino antisense oligonucleotide (MO) agent (dnd-MO). These results reported the first known and functional method of sturgeon sterilization (Linhartova et al., manuscript). We provided important information on morphology and ultrastructure of beluga spermatozoa structure by scanning and transmission electron microscopy to increase knowledge of evolutionary and taxonomic relationships among sturgeons (Linhartova et al., 2013). Finally, this thesis presents several studies with differing focus of research but with one target goal to induce germ-line chimerism in fish. All these results are prerequisite of future application and development of surrogate production in these species.
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Isolation of early stages of germ cells in pikeperch (\kur{Sander lucioperca})
GÜNGÖR, Ege
A practical technique for enzymatic dissociation and isolation of pikeperch (Sander lucioperca) (Percidae, Teleostei) early stage germ cells (eGC), including spermatogonia and spermatocytes, is reported in this study. Their potential to differentiate into functional gametes, and transmit genetic information to the next generations makes them suitable for cryopreservation and surrogate reproduction studies by germline chimera. Two different age groups (14 month old and 18 month old) of pikeperch were used to adjust the correct stage of eGC isolation. Finally the 18 month old samples were selected due to their high average gonadal volume (0.513 g). 10 ml PBS + 0.3% trypsin (304 mOsm/kg, pH 8) were used for enzymatic dissociation of testicular cells and they were sorted by centrifugation in Percoll density gradient. eGCs were identified on the basis of their ploidy level by CYSTAIN DNA 1 steps kit (PARTEC) and morphological characteristics trough by light microscopy. Cell counting was performed on histological sections and Percoll gradient layers whit the method of random square counting. The method of isolation enriched the number of eGC from 41.3% to 84.7%, obtained from the 33% of Percoll concentration.
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