Spermatogonial Stem Cell Self-Renewal Requires OCT4, a Factor Downregulated During Retinoic Acid-Induced Differentiation

The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. Octamer-4 (OCT4) plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs. Here, we show that OCT4 is required for SSC maintenance in culture and for colonization activity following cell transplantation, using lentiviral-mediated short hairpin RNA expression to knock down OCT4 in an in vitro model for SSCs ("germline stem" [GS] cells). Expression of promyelocytic leukemia zinc-finger (PLZF), a factor known to be required for SSC self-renewal, was not affected by OCT4 knockdown, suggesting that OCT4 does not function upstream of PLZF. In addition to developing a method to test specific gene function in GS cells, we demonstrate that retinoic acid (RA) triggers GS cells to shift to a differentiated, premeiotic state lacking OCT4 and PLZF expression and colonization activity. Our data support a model in which OCT4 and PLZF maintain SSCs in an undifferentiated state and RA triggers spermatogonial differentiation through the direct or indirect downregulation of OCT4 and PLZF. The current study has important implications for the future use of GS cells as an in vitro model for spermatogonial stem cell biology or as a source of embryonic stem-like cells. STEM CELLS 2008;26:2928-2937