Identification of EOMES-expressing spermatogonial stem cells and their regulation by PLZF

Long-term maintenance of spermatogenesis in mammals is supported by GDNF, an essential growth factor required for spermatogonial stem cell (SSC) self-renewal. Exploiting a transgenic GDNF overexpression model, which expands and normalizes the pool of undifferentiated spermatogonia between Plzf +/+ and Plzf lu/lu mice, we used RNAseq to identify a rare subpopulation of cells that express EOMES, a T-box transcription factor. Lineage tracing, conditional ablation, and busulfan challenge show that these are long-term SSCs that contribute to steady state spermatogenesis as well as regeneration following chemical injury. EOMES+ SSCs have a lower proliferation index than EOMES- GFRA1+ spermatogonia in wild-type but not in Plzf lu/lu mice. This comparison demonstrates that PLZF regulates their proliferative activity and suggests that EOMES+ SSCs are lost through proliferative exhaustion in Plzf lu/lu mice. Single cell RNA sequencing of EOMES+ cells from Plzf +/+ and Plzf lu/lu mice support a hierarchical model of a slow-cycling long-term SSC population supporting more rapid-cycling short-term SSCs. Overall design: 384-well plate-based 3'-end scRNA-seq was performed on two groups, Plzf +/+ and Plzf lu/lu, of cells across 4 plates. Plzf +/+ cells were spread across 2 plates and Plzf lu/lu cells were spread over 1 plate. The 4th plate contains both Plzf lu/lu (up to well C15) and Plzf +/+ (well C15 onward). Each sample in this record represents one plate.

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Sharma M, Srivastava A, Fairfield HE, Bergstrom D, Flynn WF, Braun RE