Spermatogonial Stem Cell Self Renewal Requires OCT4, A Factor Down-Regulated During Retinoic Acid Induced Differentiation

¹ Department of Pediatrics, UT Southwestern Medical Center at Dallas
² Department of Pharmacology, UT Southwestern Medical Center at Dallas
³ Department of Pharmacology; Green Center for Reproductive Biology Sciences; Howard Hughes Medical Institute; UT Southwestern Medical Center at Dallas

^* To whom correspondence should be addressed. E-mail: Christina.Dann{at}UTSouthwestern.edu.

	Abstract

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. 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 shRNA expression to knock down OCT4 in an in vitro model for SSCs (germline stem or GS cells). Expression of 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, pre-meiotic 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 down-regulation 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.