Single-cell transcriptome analyses reveal critical regulators of spermatogonial stem cell fate transitions

Spermatogonial stem cells (SSCs) are the foundation cells for continual spermatogenesis and germline regeneration in mammals. SSC activities reside in the undifferentiated spermatogonial population, and currently, the molecular identities of SSCs and their committed progenitors remain unclear.

We performed single-cell transcriptome analysis on isolated undifferentiated spermatogonia from mice to decipher the molecular signatures of SSC fate transitions. Through comprehensive analysis, we delineated the developmental trajectory and identified candidate transcription factors (TFs) involved in the fate transitions of SSCs and their progenitors in distinct states. Specifically, we characterized the A_single spermatogonial subtype marked by the expression of Eomes. Eomes⁺ cells contained enriched transplantable SSCs, and more than 90% of the cells remained in the quiescent state. Conditional deletion of Eomes in the germline did not impact steady-state spermatogenesis but enhanced SSC regeneration. Forced expression of Eomes in spermatogenic cells disrupted spermatogenesis mainly by affecting the cell cycle progression of undifferentiated spermatogonia. After injury, Eomes⁺ cells re-enter the cell cycle and divide to expand the SSC pool. Eomes⁺ cells consisted of 7 different subsets of cells at single-cell resolution, and genes enriched in glycolysis/gluconeogenesis and the PI3/Akt signaling pathway participated in the SSC regeneration process.

In this study, we explored the molecular characteristics and critical regulators of subpopulations of undifferentiated spermatogonia. The findings of the present study described a quiescent SSC subpopulation, Eomes⁺ spermatogonia, and provided a dynamic transcriptional map of SSC fate determination.