Spermatogonial stem cells (SSCs) are essential for the generation of sperm, a developmental process that occurs continuously in sexual mature males. SSCs are important clinically, as SSC defects are implicated in many cases of male infertility. In addition, there is potential to use SSCs to cure patients with defects in later stages of spermatogenesis. While progress has been made in understanding some of the fundamental characteristics of SSCs, virtually nothing is known about how SSCs are initially generated and maintained in vivo. The goal of the first Aim in our grant is to follow up on our preliminary identification of a regulatory factor, RHOX10, which has a role in SSC establishment or maintenance in vivo. When we mutated the Rhox10 gene is mice, we found it caused a dramatic loss in SSCs. During the progress period, we used this mouse mutant model coupled with a battery of approaches, including a method that examines the profile of expressed genes in single cells (SC-RNAseq), to determine the precise role of RHOX10 in SSCs. Together, our analyses ruled out that RHOX10 acts by promoting either the proliferation or survival of SSCs. Further analysis also ruled out that RHOX10 promotes the generation of SSCs by inhibiting an alternative pathway that is necessary for the “first wave” spermatogenesis, a round of sperm generation that allows for fertility at only 6 weeks of age. Instead, our data indicated that RHOX10 acts by directly promoting the differentiation of SSC precursor cells into functional SSCs. In particular, we found that RHOX10 acts on a type of SSC precursor cell called a “T1 Pro-spermatogonia [T1-ProSG],” which are non-dividing male germ cells that first emerge in the embryo and ultimately become SSCs after birth in mice. Without RHOX10, T1-ProSG accumulate in the testes of these mutant mice, as these cells are greatly impaired in their ability to proceed to the next stage of development. While we do not know precisely how RHOX10 promotes SSC establishment, we obtained evidence that RHOX10 drives the migration of precursor SSCs into the stem cell niche in the testes, providing a likely mechanism of action. During the progress period, we also defined genes regulated by RHOX10 that are candidates to function “downstream” of RHOX10 in precursor SSCs and SSCs. We also obtained evidence that RHOX10 has a second function in male germ cells – it suppresses the ability of “parasitic” DNA called “LINE1 elements” to make new copies of themselves and move randomly to new sites in the genome. If unchecked, LINE1 “jumping” (transposition) can raise havoc by disrupting existing genes. We found that not only RHOX10, but also other RHOX family members, have the ability to inhibit the expression of genes from LINE1 elements and inhibit their transposition. The second Aim of this grant was to determine whether a human regulatory factor highly related in sequence with mouse RHOX10, called RHOXF2, has similar or identical functions with RHOX10. To examine the role of RHOXF2 in its normal context, it was critical to identify conditions that allow for the survival and proliferation of normal human testicular cells. During the progress period, we made a considerable effort in doing this, using cells from human testicular biopsies that we obtained from different sources. We identified media, supplements, and surface material that were optimal for human spermatogonial growth in culture. Importantly, we also worked on the challenging problem of introducing and expressing DNA and RNA in human spermatogonia. Our evidence suggested that introduction of DNA into human spermatogonia using specific viruses (lentiviruses) is efficient, but that the RNA expressed from the introduced DNA is poorly expressed into protein. Armed with this information, we successfully depleted RHOXF2 levels in human spermatogonial cultures using a technique called “RNA interference.” To examine if human RHOXF2 has similar functions as mouse RHOX10, we performed molecular studies. These studies revealed that RHOXF2 shares with RHOX10 the ability to regulate some of the same genes, as well as the ability to suppress LINE1 retrotransposition. This provided evidence that human RHOXF2 is a “functional ortholog” of mouse RHOX10, which justified the study of mouse RHOX10 as a model system to understand regulatory mechanisms in human SSCs. Our progress on culturing human speramatogonia sets the stage for determining the biological roles of RHOXF2 in SSCs in the future.