Immortalization of mouse germ line stem cells

In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that proliferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T-antigen gene (LTAg) under the control of an ecdysone-inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazl, and specific for germ line stem cells such as Oct-4, a transcription factor, and GFRα-1, the receptor for glial cell line-derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology.