ID4 levels dictate the stem cell state in mouse spermatogonia

Abstract

Spermatogenesis is a classic model of cycling cell lineages that depend on a balance between stem cell self-renewal for continuity and the formation of progenitors as the initial step in the production of differentiated cells. The mechanisms that guide the continuum of spermatogonial stem cell (SSC) to progenitor spermatogonial transition and precise identifiers of subtypes in the process are undefined. Here we used an Id4-eGfp reporter mouse to discover that EGFP intensity is predictive of the subsets, with the ID4-EGFP Bright population being mostly, if not purely, SSCs, whereas the ID4-EGFP Dim population is in transition to the progenitor state. These subsets are also distinguishable by transcriptome signatures. Moreover, using a conditional overexpression mouse model, we found that transition from the stem cell to the immediate progenitor state requires downregulation of Id4 coincident with a major change in the transcriptome. Collectively, our results demonstrate that the level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition between the different functional states.

Retinoic acid deficiency leads to an increase in spermatogonial stem number in the neonatal mouse testis, but excess retinoic acid results in no change

“…Spermatogonial differentiation does not occur in mice treated with WIN 18,446 and consequently the testes become enriched with undifferentiated spermatogonia (Hogarth et al, 2013). Work of Helsel et al, 2017 demonstrated through functional transplantation analyses that the ID4-EGFP Bright population is essentially pure SSCs by 8 dpp. Here we found that ID4-EGFP Bright spermatogonia are enriched in the testis following WIN 18,446 treatment, suggesting an increased number of spermatogonia with stem cell potential, a possibility we confirmed by transplantation.…”

“…Treated and control cell suspensions were then analyzed for EGFP intensity using a Cytoflex flow cytometer (B53000, Beckman Coulter, CA, USA) and the CytExpert Software (Beckman Coulter) to determine the percentage of ID4-EGFP+ and ID4-EGFP- cells. Additionally within the ID4-EGFP+ cells, the percentage of ID4-EGFP Bright, Mid, and Dim cells between treatments was measured as described previously (Helsel et al, 2017). The analysis was conducted at least 3 times from 3 different treated and control donor animals to achieve technical and biological replication.…”

“…Progenitor spermatogonia are known to contain the cell machinery to respond to RA signaling and proceed through the differentiation process (de Rooij and Russell, 2000), yet there is no published evidence to indicate that the SSCs can respond to RA. The best predictor of stem cell or progenitor capacity during steady state spermatogenesis is inhibitor of DNA binding 4 (ID4) levels, particularly when trying to detect the transitional state during which SSCs transition into progenitors (Chan et al, 2014; Helsel et al, 2017). ID4 is a helix-loop-helix protein that binds other helix-loop-helix transcription factors, acting as a repressor (Benezra et al, 1990; Riechmann et al, 1994).…”

“…ID4 is a helix-loop-helix protein that binds other helix-loop-helix transcription factors, acting as a repressor (Benezra et al, 1990; Riechmann et al, 1994). Transplantation from transgenic Id4-eGfp mice demonstrated that the ID4-EGFP+ spermatogonial population contains most of the SSC population (Chan et al, 2014; Helsel et al, 2017). Further analyses of ID4-EGFP+ spermatogonia have revealed that the intensity of the EGFP signal correlates with regenerative capacity, with the ID4-GFP Bright population being pure SSCs (Helsel et al, 2017).…”

Spermatogonial stem cells and spermatogenesis in mice, monkeys and men

“…3A). Based on the restricted pattern of expression, some have suggested that cells expressing ID4, PAX7 and/or BMI1 might be the ultimate spermatogonial stem cells (SSC ultimate ) (Helsel et al, 2017; Lord & Oatley, 2017; de Rooij, 2017). Indeed, the expression of each marker on functional stem cells has been confirmed by SSC transplantation and/or lineage tracing.…”