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

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. 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.

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

“…Vimentin, an intermediate filament of Sertoli cell cytoskeleton, plays an important role in maintenance of spermatogenesis in the testes [53]. Using qPCR, we further confirmed the expression of mRNA encoding other SSC specific markers (viz., β1 integrin and Oct-4) required for colonization and self renewal of SSCs [54,55]. A relatively higher abundance of vimentin mRNA observed in the study might be due to the fact that most SSC cultures contain a mixture of testicular cells constituting as less as 1.33 % SSCs and other testicular cells mainly comprising of Sertoli cells [26].…”

Crucial Genes and Pathways in Chicken Germ Stem Cell Differentiation

“…We identified thousands of differentially expressed genes (DEGs) 4 in this process, and from these we chose 173 candidate genes, including 98 genes involved in cell differentiation, 19 involved in the metabolic process, 56 genes involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, PSMD7, and so on. In addition, we found that there were 18 key signaling pathways mainly involved in cell proliferation, differentiation, and signal transduction process like TGF-␤, Notch, and Jak-STAT.…”

Pluripotency of Male Germline Stem Cells

“…Although the expression level is not as high as in pluripotent stem cells, Oct4 knockdown has detrimental effects on the selfrenewal and proliferation of SSCs (Dann et al, 2008). The mechanism of Oct4 regulation of SSC self-renewal appears to be independent of the Plzf-mediated pathway.…”

“…Accordingly, mouse SSCs express several pluripotency marker genes, including 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-myc), although the OCT4 and SOX2 protein expression levels are low or absent due to transcriptional repression and possibly translational inhibition (Kanatsu- Shinohara et al, 2008;Oatley et al, 2006). A low level of OCT4 expression is required for SSCs to function properly because OCT4 knockdown negatively affects SSC maintenance (Dann et al, 2008). Sox2 is expressed transcriptionally in mouse SSCs, but its protein level and functions have not yet been explicitly determined.…”