The Effects Of Autophagy On The Biplogical Characteristics Of Male Germline Stem Cells Of Dairy Goat

Male germline stem cells（m GSCs）, also known as Spermatogonial stem cells（SSCs）, is one kind of undifferentiated male germ cells and located in the basement membrane of seminiferous tubules. Sperms are produced by m GSCs differentiating into spermatocytes. m GSCs, as the only sort of adult stem cell that can pass on their genetic materials to offspring, has self-renewal ability and multilineage differentiation potential. At present, the study of m GSCs mainly focus on the isolation and culturing of m GSCs from different species, and on the mechanism of maintaining self-renewal ability, and on the function of dedifferentiation and transdifferentiation of m GSCs in translational medicine studies. Moreover, the studies about self-renewal mechanism concentrate mainly on the regulatory effects of cytokines and transcription factors on self-renewal.Autophagy is put forward by Ashford and Porter in 1962, after the discovery of the phenomenon “self-eating” in hepatic cell. Autophagy is responsible to degrade dysfunctional organelles and redundant proteins for the reconstruction of cells in many cellular events, such as metamorphosis, ageing and differentiation. At present, the research of autophagy has made great progress, but most of it centered on developmental mechanism, tumour and ageing. In recent years, more and more researchers concentrate on the function of autophagy on reproduction. So in this study,we focused on autophagy and its function on the biological characteristics of male germ cells and primarily explored the relationship of autophagy to the self-renewal and differential ability of m GSCs. This study laid a foundation for insight into the mechanism of spermatogenesis comprehensively.In this study, we use Dairy Goat as animal model. At first, we screened and got the autophagy trace tool（Gm GSCs-I-SB-EGFP-LC3 B, expressing fused protein EGFP-LC3 B stably） from Gm GSCs-I-SB, which was got by immortalizing the primary Dairy Goat male germline stem cells, to ensure the existence of autophagy in Gm GSCs-I-SB and that Gm GSCs-I-SB-EGFP-LC3 B can be a real time monitor of autophagy in Gm GSCs-I-SB. Next, by using the Rapamycin and Chloroquine to promote or inhibit the autophagic development of Gm GSCs-I-SB, we detected the change of cell cycle, apoptosis and self-renewal markers. On the other hand, we observed the number of autophagosomes was remarkablely increased in the differentiating Gm GSCs-I-SB-EGFP-LC3 B.1.Establishment of autophagy trace tool—Gm GSCs-I-SB-EGFP-LC3BAt first, we ensured the phenomenon that “self-eating” exists in the Gm GSCs-I-SB by Immunofluorescence staining, Western blotting, Transmission electron microscope. Second, we design a pair of primers based on the known sequences U55763.1 and XM₀11529085.1 in NCBI to amplify the fragment that can expressing EGFP-LC3 B fused protein from the p EGFP-C1-LC3 B vector and then inserted this fragment into lentiviral vector p TRIP-CAGG-puro. The results of double digestion by Xba1 and Bam Hl restriction enzymes and sequencing indicated that we reconstructed the lentiviral vector p TRIP-CAGG-EGFP-LC3B-puro successfully. Further, Gm GSCs-I-SB-EGFP-LC3 B was screened by Puromycin after Gm GSCs-I-SB was transducted with reconstructed lentiviral virus solution—p TRIP-CAGG-EGFP-LC3B-puro. At the 10 th generation, we accounted the numbers of EFGP-positive cells of Gm GSCs-I-SB-EGFP-LC3 B with Flow cytometry, and found that green fluorescence of Gm GSCs-I-SB-EGFP-LC3 B can co-locate with red fluorescence of LC3 B by Immunofluorescence staining, and EGFP-LC3 B was also detected by Western blotting about 42 KDa. After got this stably expressing EGFP-LC3 B cell line, we used non-serum starvation, Rapamycin and Chloroquine to stimulate Gm GSCs-I-SB-EGFP-LC3 B and observed the occurrence of green autophagosomes with EGFP tag. All the results indicated that Gm GSCs-I-SB-EGFP-LC3 B can be used to monitor the development of autophagy of Gm GSCs-I-SB.2. The relationship between autophagy and m GSCs.At first, we chose Rapamycin（0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nmol/L） and Chloroquine（0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μmol/L） with different concentration to stimulate Gm GSCs-I-SB, and then selected 100 nmol/L Rapamycin and 100μmol/L Chloroquine as the safe concentration that can not make effects on cell viability, but can effect the development of autophagy. After incubated with indicated concentration stimulus for 24 h, the proliferation was down-regulated and the level of apoptosis was up-regulated in Chloroquine group, but self-renewal marker Plzf was up-reguleted in Rapamycin group. Besides, we observed the occurrence of autophagy in Gm GSCs-I-SB-EGFP-LC3 B in the spontaneous differentiation process from embryonic bodies and the inducement of neural-like cells by monolayer culture.