PROJECT SUMMARY/ABSTRACT Most adult tissues are maintained by resident adult stem cells that maintain the function and integrity of the tissue. As old cells die or are damaged, new cells are produced from adult stem cells. In many tissues there is emerging data suggesting the presence of both rapid-cycling and quiescent stem cells. Rapid cycling cells are actively engaged in tissue repair while slow-cycling, or G0-arrested cells, are reserve cells. Recently published work, and preliminary studies in this proposal, support the hypothesis that spermatogonial stem cells (SSCs) in the testis are heterogeneous in their cycling status and that disruption of the normal cell cycle can interfere with both self-renewal and differentiation. We have identified a subpopulation of spermatogonia that express EOMES, a T box transcription factor. Using lineage tracing we have shown that they contribute to steady-state spermatogenesis and to regeneration following germ cell ablation by busulfan. In Plzf mutant mice, which show an age-dependent depletion of SSCs, EOMES+ cells cycle more rapidly, suggesting that age-dependent depletion of SSCs is caused by proliferative exhaustion. The central hypothesis of this proposal is that spermatogonial stem cells (SSCs) are also heterogeneous with respect to their cycling status and that there are both rapid cycling and slow cycling SSCs. We propose that proper regulation of the cell cycle is critical for maintaining the homeostatic balance between self-renewal and differentiation and that loss of cell cycle regulation can lead to age-dependent loss of SSCs due to the mis-regulation of critical self-renewal genes. In Specific Aim 1 we will quantify the frequency of cell cycle asynchrony, the concordance of SSC marker expression and cycling status, and the effect of mutation of Plzf on both of these parameters. In Specific Aim 2 we will utilize an allele of Ki67 (Mki67-RFP) to flow sort cycling and non-cycling cells from the larger pool of GFRA1+ population. Single cell RNA sequencing (scRNAseq), and single nuclei Assay for Transposase Accessible Chromatin (snATACseq) of the cycling and non-cycling cells will provide and independent assessment of the non-cycling G0 population of SSCs, whether Eomes and other markers of SSCs cells are enriched within the population, and whether there is a previously unidentified population of G0 cells within the SSC pool. Lastly, In Specific Aim 3 we will assess how mutation of Batf, a regulator of Eomes expression, enhances the germ cell loss phenotype in Plzf lu/lu mutants, and assess the role of the mis-regulation of cell cycle genes in the driving the hyperproliferative phenotype.