Conservation of a gene expression barcode that defines spermatogonial stem cells in mice and humans




Singh, Anukriti

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Spermatogonial stem cells (SSCs) are undifferentiated spermatogonia that sustain mammalian spermatogenesis by producing progeny that will either retain stemness (self-renew) or become progenitors that are committed to differentiation. The mechanisms that drive these alternate fates remain poorly understood partly because 1) SSCs are rare, 2) undifferentiated spermatogonia (including SSCs) are heterogeneous, and, 3) SSCs are problematic to prospectively distinguished from progenitors. We reasoned that single-cell transcriptomes of cells highly enriched for mouse SSCs could help identify a gene expression signature characteristic of SSCs and conserved among some human spermatogonia. To this end, we performed single-cell RNA-seq on 134 postnatal days 6 (P6) and 151 adult ID4-EGFP+ spermatogonia and subdivided these cells based on intensity of EGFP epifluorescence into EGFP-bright (pure SSCs) and EGFP-dim (progenitors), which matches their functional distinctions based on transplantation (Helsel et. al. 2017). Thousands of genes were differentially-expressed between EGFP-bright and dim subpopulations at both stages, including a subset which were conserved across postnatal development. While EGFP-bright and dim subpopulations were heterogeneous in their gene expression profiles, they were phenotypically separable by 266 differentially expressed genes (p<0.05, ≥2-fold change) that constitute a putative mouse SSC barcode.

Among genes that were upregulated in EGFP-bright (SSCs) were components of the cellular response to GDNF (Gfra1, Ret, Tcl1, Etv5, Fos) and FGFs (e.g., Dusp1, Dusp6). In EGFP-dim (progenitors), genes involved in the regulation of translation (e.g., Eif4ebp1), retinoic acid biosynthesis (Rbp1), activation of retinoic acid receptors and pyrimidine biosynthesis (Upp1) were enhanced. In addition, we compared the mouse SSC barcode to single-cell transcriptomes from 323 adult human undifferentiated spermatogonia (isolated from 9 individuals) which were stratified based on levels of ID4 expression. The highest ID4-expressing neonatal mouse, adult mouse, and adult human spermatogonia exhibited significant conservation of this gene expression barcode (27 genes - p<0.05, ≥2-fold change; 327 genes – p<0.05, ≥1.5-fold change). Collectively, these findings point to the first putative gene expression signature (barcode) of mouse SSCs and key components of this signature that are conserved between mouse and human spermatogonia. Ultimately, these data, for the first time, reveal the identity and phenotype of human SSCs.


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Integrative Biology