Mechanisms of Specification and Maintenance of Foundational Spermatogonial Stem Cells in Mice

The continuous production of sperm throughout the male reproductive lifespan is sustained by spermatogonial stem cells (SSCs). During fetal and early postnatal (P) mouse testis development, only a subset of prospermatogonia become SSCs, but how these cells are specified and maintained is poorly understood. Single-cell RNA-Seq data was used to define genes with differential mRNA levels between SSC and non-SSCs (progenitor, differentiating spermatogonia) that may distinguish the origin of SSCs during germline development. Indeed, a subpopulation of embryonic day (E) 16.5 prospermatogonia contains mRNAs from a subset of genes expressed in SSCs we termed the 'SSC signature' (SSCSig). We found this signature is also present in prospermatogonial subpopulations at E13.5-E16.5, which correlates with cell cycle activation but was absent at E18.5. S-phase lineage tracing demonstrated a subset of prospermatogonia at P0 comprising the nascent SSCs were preferentially labeled with EdU at E13.5-E14.5, suggesting they were destined to become SSCs. To elucidate the mechanisms that maintain this gene expression distinction between SSCs and non-SSCs, we tested whether the mRNA levels characteristic of the SSCSig arise from differential transcription or transcript stability. Using metabolic labeling of nascent transcripts, we found that representative SSC genes were transcribed significantly more robustly than non-SSC genes, while there was no significant difference in the rates of transcript degradation. Overall, our results suggest nascent SSCs arise preferentially from a subpopulation of dividing prospermatogonia at E13.5-E14, and the mRNA levels characteristic of the postnatal SSC gene expression signature are regulated by differential transcription.