Spermatogonial stem cells (SSCs) are self-renewing cells essential for adult spermatogenesis that hold promise for treating male infertility. In this proposal we focus on two topics revolving around SSCs. First, we will identify “transcription factor (TF) circuits” critical for SSC establishment. Identifying such TF circuits will allow us to ultimately define a functionally defined (not merely “omics” defined) TF network that drives immature germ cells to become stem cells. Second, we will follow-up on our surprising finding that knockout (KO) of a transcription factor important for spermatogenesis causes expansion of SSCs. Understanding the underlying mechanism for this potential compensatory response to genetic insult has the potential to reveal insights into how human SSCs respond to toxic insults and infertility. The molecular mechanisms by which SSCs are initial generated in vivo are poorly understood. One of the few proteins that have been shown to drive SSC establishment is the homeobox TF, RHOX10, which we showed promotes SSC precursor cell differentiation and the initial seeding of SSCs in the seminiferous epithelium. Given that TFs are regulatory factors that control batteries of downstream genes, our discovery that RHOX10 drives SSC establishment provided an opportunity to define key genes critical for this process. Indeed, we recently identified scores of direct RHOX10-target genes, including a remarkably large number of TF genes with known roles in SSCs. This led us to hypothesize that a TF network is critical for SSC establishment, which we propose to test. This is critical, as even though other studies have identified hundreds of downstream genes regulated by TFs at various germ cell stages, it has not been ascertained which of these downstream genes are functionally important for the action of a given TF. In Specific Aim 1, we will perform functional experiments to determine the regulatory relationships of TFs, thereby allowing us to define functional TF circuits. As evidence of feasibility, we recently defined—through rescue experiments—3 TFs that function in a TF circuit to drive SSC precursor cell differentiation. The other focus of this proposal revolves around our unexpected recent discovery that adult Rhox10-null mice have expanded numbers of SSCs. Understanding the mechanisms that drive SSC expansion is critical for devising approaches to treat infertility. Our preliminary data support 3 testable models to explain the SSC expansion in these KO mice, including a compensatory mechanism model in which SSCs undergo proliferative expansion when they sense downstream spermatogenic defects. In support of such a feedback mechanism operating in humans, it was recently reported that cryptozoospermia infertility patients have a selective and robust increase in the number of SSCs, as defined by single-cell RNAseq analysis. In Specific Aim 2, we will determine the cellular and molecular mechanisms responsible for the expansion of SSCs i...