To Explore The Potential Of Pluripotency Factor NANOG In Mouse Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs),derived from primordial germ cells(PGCs),are the only germline stem cells in male seminiferous tubule of testis.SSCs have the ability of self-renewing,differentiating into all types of germ cells and generating sperms that carrying genetic information to offspring.Shinohara' group in 2004 showed that few pluripotent stem cells could derive from p53-deficient testes of new born mice.Furthermore,Guan et al in 2006 established cultures of pluripotent mouse adult germline stem cells(ma GSCs)from 4-6 weeks old mouse testis.Seandel et al in 2007 also successfully cultured multipotent adult spermatogonial derived stem cells(maSCs)from over one year old mouse testis.Again,Ko et al in 2009 established germline-derived pluripotent stem cell(gPS)culture in vitro using 5-8 weeks mouse testis.Of those studies,however,all showed an extremely low efficiency of derivation of pluripotent stem cells from SSCs.By comparing transcriptional levels of pluripotency genes among embryonic stem cells(ESCs),induced pluripotent stem cells(IPS cells)and SSCs,Brinster et al found that pluripotency factor Nanog were highly expressed in ESCs and IPS cells but undetectable in SSCs.Recently,Toppari et al reported that a few NANOG positive cells existing in certain stages of mouse seminiferous tubules,may represent a small portion of undifferentiated spermatogonia with the ability of potency conversion in mouse testis.Therefore the scientific question in my paper is: are those NANOG positive cells representing the population of SSCs with pluripotency?Firstly,we characterized the expression of Nanog in the testis and SSCs in mice.We found low level of Nanog expression in mouse testis but unable to find the expression of Nanog in SSCs.Immunohistochemistry showed that the expression of NANOG in the basement membrane of mutiple stages.We isolated spermatocyte?round spermatid by STA-PUT,and found Nanog levels is similar to SSCs.Because Nanog is absent in SSCs,we generated letivirus-mediated Nanog overexpression plasmids and transfected them into cultured mouse SSCs.Overexpression of Nanog was validated at mRNA levels by quantitative real-time PCR(qRT-PCR)and at protein levels by Western Blotting.Though cells had a modest increase on proliferation.Gene expression analysis showed that SSCs marker,Stem cell marker and cell cycle?differentiation marker had no significant difference in the control and Nanog overexpressed SSCs.However cell apoptosis gene Bax was down-regulated in SSCs when Nanog overexpressed.We next transplanted control SSCs?SSCs with NANOG overexpression,?IPS cells into mouse testes.Two months later,we found that control SSCs?SSCs with Nanog overexpression did not produce any tumor-like tissue or teratoma in recipient mouse testis.As a control,IPS cells transplanted could develop into teratoma tissues in recipient mouse testes.X-gal staining further showed that SSCs with Nanog overexpression generated normal spermatogenesis with no visible difference in the number of regeneration colonies,comparable to colonies derived from control transplantation.Collectively,our preliminary data indicated a single induction of pluripotent factor Nanog did not converse the potency of SSCs.Nevertheless,our study provided a foundation for future exploration of mechanism in determining SSC potency.