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The Mechanism of GDNF Mediates Self-renewal of Dairy Goat Male Germ-line Stem Cells

Author: SunJunWei
Tutor: HuaJinLian
School: Northwest University of Science and Technology
Course: Animal Biotechnology
Keywords: Male germ stem cells GDNF Signaling pathway Dairy goat
CLC: S827
Type: Master's thesis
Year: 2011
Downloads: 36
Quote: 0
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Male germline stem cells (male germ-line stem cells, mGSCs), also known as spermatogonial stem cells (SSCs), biology research in recent years, the most active, one of the most attractive areas of the male germ stem cell research in the livestock production, biomedical and other fields have a wide range of applications. Glial cell line-derived neurotrophic factor (glial cell line-derived neurotrophic factor, GDNF) is a growth factor of the first found the regulation mGSCs self updated. Studies have shown that, GDNF is the regulation mGSCs self-renewal in vitro most basic growth factor, and its role in a dose-dependent manner. Mouse male germ stem cell lines of GDNF on mGSCs regulation of self-renewal through PI3K/Akt, Ras/Erk1/2, Src family kinase signaling pathway to achieve, but for different species, different cell lines, GDNF-mediated signaling pathway but not the same, at the same time whether the regulation of GDNF on the proliferation of cattle, goats and other animals mGSCs depends on several of these signaling pathways have not been reported. We examined the expression of GDNF family receptor milk the goat testicular tissue in different periods, further separation and purification of the dairy goat mGSCs its germ cells and pluripotent cell-specific surface antigen detection. Then using different concentrations of GDNF (0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL) to cultured dairy goat mGSCs screened maintain optimal GDNF concentration the dairy goat mGSCs self-renewal. Finally, in containing GDNF milk goats mGSCs the culture was added, a specific inhibitor of PI3K and MEK initial screening of GDNF role in the dairy goat mGSCs the main signal path. Of GDNF family receptor GFRa1 the means of expression using RT-PCR and immunohistochemistry GDNF family receptor GFRa1 in the different age periods of fetal and newborn sheep, adolescent and adult dairy goat testicular tissue expression . RT-PCR results show strong the GFRa1 expression in the fetal period, its expression level decreased gradually with increasing age, while the immunohistochemical results displayed GFRa1 only expression in adult testis tissue. This shows GFRa1 dairy goat testicular tissue expression, but the expression of the law may be the same little mouse and human. 2. Of milk goats mGSCs the separation, purification and identification of hybrid enzymes (collagenase 2 mg / mL DNA enzyme 20μg/mL the decomposition 2 mg / mL) enzyme to digest dairy goat testicular tissue, compared fibronectin differential 2 h paste cell wall and not adherent cell alkaline phosphatase (AP) staining and found that the differential speed 2 h non-adherent cells were formed by cloning AP staining coloring darker, and the whole cloning coloring adherent cells 2 h cloning presents part of the AP-positive, the differential 2 h nonadherent cells mGSCs higher levels. Differential 2 h nonadherent cells, germ cells and pluripotent cell surface the antigen (GFRa1-CD49f Stra8, Vasa, TERT, Oct4) immunofluorescence staining and found that these cells showed a positive, relatively pure. Goat's milk mGSCs. Screening of GDNF optimum concentration containing different concentrations of GDNF (0 ng / mL, 5 ng / mL and 10 ng / mL, 20 ng / mL) of milk goats mGSCs the culture the dairy goat mGSCs fluid cultures purified, by the groups doubling time, cell proliferation (PCNA, BrdU immunofluorescence staining) and expression of pluripotency analysis showed that 10 ng / mL GDNF group training mGSCs its population doubling time than other groups short, proliferative capacity was significantly stronger, and more no significant differences in gene expression can, in order to determine the the updated optimal concentration of 10 ng / mL GDNF to maintain milk goats mGSCs self. 4. GDNF-mediated signaling pathway study used dairy goat mGSCs-control culture medium containing 10 ng / mL GDNF culture medium containing 10 ng / mL GDNF 10μmol / L PI3K inhibitors and containing 10 ng / mL of GDNF 10μmol / L MEK inhibition the agent were cultured with purified milk goats mGSCs, population doubling time, cell proliferation, pluripotency gene expression, as well as by RT-PCR, fluorescence quantitative PCR Western Blotting and cell cycle analysis showed that the GDNF by PI3K / Akt and MEK/ERK1/2 two signal pathway in dairy goats mGSCs self-renewal, and found that GDNF may mainly work through the PI3K/Akt signaling pathway.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Goat
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