Project Summary We are requesting funds to purchase a qPCR machine, a cell culture incubator, and a cryostat, for the purpose of studying the molecular mechanisms underlying spermatogonial stem cell (SSC) establishment, self-renewal, and differentiation. SSCs are self-renewing stem cells essential for adult spermatogenesis that hold promise for treating male infertility, a condition affecting >200 million men world-wide. The parental NIH grant is focused on experiments to better understand SSCs as a foundation to develop such SSC therapy. The 3 items requested—a qPCR machine, a cell culture incubator, and a cryostat—are all essential for these studies. First, the primary goal of Aim 1 of our NIH grant is to test the hypothesis that transcription factor (TF) circuits are critical for the initial generation of SSC from SSC precursor cells (called prospermatogonia [ProSG]). All 3 pieces of equipment requested will be essential to test this hypothesis. qPCR analysis will measure the expression of germ cell stage-specific gene markers as a means to determine ProSG and SSC frequencies in all 3 Subaims in Aim 1. For example, qPCR analysis will be used on the different transgenic mouse models we are employing to test our primary hypothesis in Subaim 1.1 – that a RHOX10-DMRT1 TF circuit drives SSC establishment in vivo. The cryostat will be used to make sections from dissected testes from these transgenic mouse strains to provide single-cell information on the specific germ cell subsets in vivo. Immunofluorescence (IF) analysis will be used on these sections to assess the expression of ProSG and SSC protein markers at the single-cell level. The cell culture incubator will be used to perform the experiments in Subaims 1.2 and 1.3 described in the parental NIH grant, which are focused on defining TF cascades in cultured ProSG and SSCs in vitro. Culturing these ProSG and SSCs will require a dedicated cell culture incubator, set at 34oC to mimic the temperature of the testis. Extensive qPCR analysis will also be required for these Subaim 1.2 and 1.3 experiments. Second, we are testing 3 models (delineated in Aim 2 of our parent grant) that explain our surprising finding that the loss of the RHOX10 TF causes expansion of SSCs in adult testes. All 3 pieces of equipment requested are critical to test these models, following the same approaches, as described above. Third, we are studying the conserved role of the Activin A and AKT pathways in both human and mouse SSCs in vitro, which will require the requested qPCR machine and cell culture incubator. Fourth, we are defining and characterizing novel protein markers labeling different human spermatogonial subsets harboring SSCs, which will require a cryostat for IF analysis. Finally, we are studying how the nonsense-mediated RNA decay (NMD) pathway influences SSCs and other stages of spermatogenesis, a study that requires all 3 requested equipment items.