Erişkin fare ovaryumunda olası germline ve pluripotent kök hücrelerin araştırılması
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Date
2015
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Biyoteknoloji Enstitüsü
Abstract
According to the classical knowledge of biology, in the ovary of female mammals there are a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in the last 10 years, the results of studies about renewal and formation of oocytes and follicles in the adult mammalian ovary are lead to changing of this view completely. The aim of this thesis is demonstration of presence of putative germline and pluripotent stem cells and investigates the possibility of neo-oogenesis in the adult mouse (8 week-old BALB/c) ovary. Immunoflorescence staining of ovarian tissue sections revealed the presence of pluripotent stem cell (Oct-4, Nanog and Sox2) and germline stem cell (DAZL and DDX4/VASA) markers in the cytoplasm and nucleus of oocytes in different follicular stages. Presence of these proteins was also observed in the nucleus and cytoplasm of the cells that harvested from ovarian cell culture by immunoflorescence staining. In the ovary cell culture, oocyte differentiation process led to formation of morphologically oocyte-like cells and in some of these cells parthenogenesis was occurred. As a result of differentiation process formation of follicle-like structures was also observed. In the ovarian tissue and non-differentiated cells that harvested from cell culture, expression of the Oct-4, Sox2, Nanog, DDX4 and DAZL genes was demonstrated by qRT-PCR method. Nevertheless, in the harvested cells comparing ovarian tissue, despite the increased expression of Nanog, expression of Oct-4, Sox2, DAZL and DDX4 was decreased significantly (p<0.05). In the cells that differentiated into oocyte, expression of oocyte specific genes and in the parthenogenesis occurred cells, expression of pluripotent specific genes was demonstrated by RT-PCR method. As a result of western blot analysis, expression of Oct-4, Sox2, Nanog, DDX4 and DAZL proteins was observed in the ovarian tissue and non-differentiated cells that harvested from cell culture. Based on our study outputs, presence of pluripotent and germline stem cells in the adult mouse ovary was demonstrated. Under in vitro circumstances, differentiation potential of these cells into oocyte-like cells, follicle-like structures and in some cases parthenogenesis-like phenomena was observed.
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Biyoteknoloji