Spermatogonial stem cells and progenitors are refractory to reprogramming to pluripotency by the transcription factors Oct3/4, c-Myc, Sox2 and Klf4

Abstract

The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events.

EZHIP constrains Polycomb Repressive Complex 2 activity in germ cells

“…We first investigated the expression of Ezhip during spermatogenesis in the different subpopulations of germ cells sorted from adult mice based on staining for α6-integrin, the tyrosine kinase receptor c-Kit, and DNA content, as previously described 41,42 . Ezhip was mostly expressed in spermatogonia (α6-integrin positive, Supplementary Figure 5E).…”

“…Testicular single-cell suspensions were prepared from 2-3-months-old from WT and Ezhip -/mice as described previously 41 . The albuginea was removed and the seminiferous tubules were dissociated using enzymatic digestion by collagenase type I at 100 U/ml for 25 minutes at 32°C in Hanks' balanced salt solution (HBSS) supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO4, 1.3 mM CaCl2, 6.6 mM sodium pyruvate, 0.05% lactate.…”

Immortalized murine fibroblast cell lines are refractory to reprogramming to pluripotent state

“…Activation of AMP-activated protein kinase (AMPK) provides a metabolic barrier to reprogramming somatic cells into stem cells [ 48 , 53 , 54 ]. Another relevant example is spermatogonial stem cells (SSCs) which are refractory to reprogramming to pluripotency [ 55 ]. Compared to ESCs, SSCs express lower levels of Oct4 and Sox2, and higher levels of Klf4 and cMyc.…”

“…Deregulated expression of these pluripotency genes triggered by ectopic overexpression of OSKM and abnormal epigenetic regulation of specific regulatory elements involved in reprogramming might be the cause of the refractoriness of these cells to iPSC reprogramming. Moreover, it was shown that p53 pathway does not contribute to the refractoriness of SSCs to reprogramming [ 55 ]. It was also shown that naked mole rat (NMR) fibroblasts have drastically reduced propensity for reprogramming, compared to mouse fibroblasts.…”

Frequency and Markers of Precursor Lesions and Implications for the Pathogenesis of Testicular Germ Cell Tumors

“…However, in a study of adult mice, a panel of transcription factors did not induce a pluripotent pattern in germ cells. 36 The panel of transcription factors might also be the background for the seminomatous immunophenotype in the malignant germ cells of precursor lesions. GCNIS and SEM express SOX17 but not SOX2, whereas EC expresses SOX2 but not SOX17.…”