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Stem cells in somatic tissues of mammals - past and present view
Hlavatá, Adéla ; Kořínek, Vladimír (advisor) ; Krylov, Vladimír (referee)
The homeostatic self-renewal of tissues in the adult mammal organism is maintained by stem- cell activity. The majority of tissue-specific stem cells are dispersed in a tissue in a low number. The small intestinal epithelium is a suitable model tissue for study of the stem cells because of its regular structure and rapid self-renewal. One of the first knowledge about the intestinal stem cell characteristics was obtained from the experiments with the mice embryonal aggregation chimeras and the transgenic chimeras. There were obtained a crucial role of the Wnt signalization pathway in the control of dynamics of the stem cells and contextual coherent finding of the unique molecular marker protein of the intestinal stem cells Lgr5. Subsequently, the genetically modified mice with "reporter" genes produced from the Lgr5 locus became the main tool in the intestinal epithelial stem cell research. The views on dividing organization and particular identity of the intestinal stem cells are still controversial. keywords: stem cells, crypt base cells, intestinal epithelium, chimera, the Wnt signaling pathway, Lgr5, reporter mouse strains
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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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Location of Balbani cytoplasm in sturgeon eggs as primordial gonocytes precursor during fertilization
DVOŘÁK, Matěj
The oocytes of many organisms, including fish, contain germ plasm. Germ plasm is maternally inherited determinants of germ cells. New-found germ plasm has been also described in previtellogenic sturgeon oocytes as Balbani cytoplasm. It contains precursors of germinal granules so called nuage. Germinal granules have been detected as a part of primordial gonocytes (PGCs) and according to some studies it play the role in the formation PGCs. PGCs are embryonic cells from which the gametes are differentiated. These cells has potential to transmit genetic information to the next generation. PGCs or subsequently spermatogonia therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ?line chimeras. Nevertheless the first step for successfully transplantation is visualization of PGCs. It is usually done using injection of GFP (green fluorescent protein) mRNA conjugated with mRNA, such as vasa and nanos, to the area of egg with PGC precursor localization (nuage) early after fertilization. Electron microscopic observation at this study revealed three main principal layers (a topmost alveolar layer, a zona radiata externa and a downmost layer as an zona radiata interna), a layer of cortical granules in unfertilized eggs and a perivitelline space in fertilized eggs; then germ plasm, containing nuage, which was dispersed only in the vegetal pole of ovulated sturgeon eggs. In addition, we observed formation interspace between the zona radiata externa and zona radiata interna. Sturgeon eggs have too strong envelopes for injection by glass needle. The glass capillary needle penetration could be facilitated by separation of parts of the egg envelopes as the alveolar layer and the zona radiata externa from interspace, which created by separation of the zona radiata externa and zona radiate interna by tweezers.
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