New Regulators of Spermatogonial Stem Cells: RHOX Homeobox Transcription Factors
Infertility afflicts a remarkably high percentage (~15%) of couples, with male factor defects being responsible for more than ½ of these cases. One-third of these male infertility cases have no known cause. For most infertile men, the only “treatment” is in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), both of which are costly, invasive for the female, and yield low pregnancy rates and even lower live birth rates. There is also evidence that the offspring born of IVF and ICSI procedures are at greater risk for developing a variety of conditions, including diabetes, obesity, and chromosome and epigenetic aberrations. Thus, there is a great need for alternative approaches, many of which revolve around knowing more about the stem cells in the testis—the spermatogonial stem cells (SSCs)—as they are essential for giving rise to sperm. Towards this goal, we have identified a regulatory gene that we have considerable evidence promotes the self-renewal of SSCs. In this application, we propose to elucidate the precise biological functions and molecular targets of this regulatory gene in SSCs. To determine its role in humans, we will use molecular approaches to knock down its expression in SSCs derived from human testicular biopsy samples. By determining the underlying mechanisms by which human SSCs self-renew, this research has the potential to generate novel therapies for male infertility, including for cancer patients rendered infertile due to chemotherapy.
Fertility defects are extremely common in human males. At least 7% of men of reproductive age in the U.S. are either infertile or subfertile. This translates to over 1 million men in California suffering from major fertility deficiencies. In contrast to women, only a very limited number of men with primary infertility have a medically treatable condition. Rather than direct treatment, male infertility is typically dealt with by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Both of these procedures are associated with several risks, including chromosomal and epigenetic abnormalities, and they yield low pregnancy rates and even lower live birth rates. They are typically not covered by insurance in California and are extremely costly—$15,000 to $20,000 for a single cycle—and most couples undergo more than one cycle. In total, it is estimated that more than $40,000,000 million a year is spent on these procedures in California alone. This application is focused on new approaches for treating male infertility that revolve around using spermatogonial stem cells (SSCs). In addition to providing potential cures for idiopathic male infertility (~32% of male infertility cases in California), SSCs provide a source of germ cells for engendering fertility to patients undergoing cancer chemotherapy treatment. By expanding SSCs cryopreserved before such patients undergo chemotherapy, their fertility can later be restored by transplantation.
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 an interest in using SSCs to cure patients with defects in later stages of spermatogenesis. While some progress has been made in understanding some of the fundamental characteristics of SSCs, how SSCs are initially generated and maintained in vivo remains poorly understood. We have identified a transcription factor, RHOX10, that our studies have suggested has roles in SSCs. When the Rhox10 gene is mutated in mice, this causes phenotypic defects consistent with a dramatic loss in SSCs. During the progress period, we used this mouse mutant model coupled with a battery of approaches to examine whether indeed RHOX10 is required for the normal accumulation of SSCs. Using a germ cell transplantation assay, which is regarded as the gold standard for assaying SSC numbers, we found that Rhox10-knockout (KO) mice have dramatically lower numbers of SSCs than wild-type control mice. This SSC defect was observed at an early postnatal age, raising the possibility that RHOX10 is important for the initial establishment of SSCs, a developmental step that occurs soon after birth in mice. In further support of a dramatic reduction in SSCs, we found that both postnatal and adult Rhox10-KO mice have reduced numbers of testicular cells expressing markers known to be expressed on SSCs. In principal, the reduction in SSCs could be caused by an embryonic defect, such as in the formation or expansion of primordial germ cells in the embryo. We found that this is unlikely to be the case, as we observed normal numbers of germ cells in the testis of Rhox10-KO mice at late points of embryonic development and at birth. We then took a series of approaches to assess how Rhox10 functions to drive the initial establishment of SSCs, a developmental step that occurs postnatally. Assessment of proliferation and apoptosis markers demonstrated that there was no measurable defect in the proliferation or survival of SSCs or the germ cells that give rise to them: pro-spermatogonia (Pro-SG). After ruling out these and other possibilities, we considered that Rhox10 acts instead by promoting the differentiation of Pro-SG into SSCs. To test this, we took advantage of markers relatively specific for Pro-SG and SSCs, coupled with reporter mice and double-immunofluorescence analysis. Together, the data we obtained provided strong support for the notion that, indeed, Rhox10 is necessary for the efficient conversion of Pro-SG into SSCs. To explore this in more depth, we used a new genome wide approach – single cell-RNAseq analysis. Comparison of single germ cells from early postnatal Rhox10-KO and control mice confirmed that loss of Rhox10 causes a defect in the progression of Pro-SG into SSCs. Single cell-RNAseq analysis also identified putative Rhox10 target genes in specific germ cell subsets, including in Pro-SG and SSCs. Finally, this approach identified many transcripts highly enriched in specific wild-type and Rhox10-null germ cell subsets that are candidates to serve as Pro-SG- and SSC-specific markers. This is a critical advancement for the field, as currently no such markers exist. Given that Pro-SG are considered to be the immediate precursor cells of SSCs, having markers that define them will be broadly useful for future studies on this critical developmental step. SSC-specific markers will have major utility for both basic science research and for future clinical applications of SSCs. In conclusion, during the last progress period, we obtained definitive evidence that Rhox10 is critical for the initial generation of mouse SSCs. We obtained a wealth of evidence that RHOX10 promotes the differentiation of SSC precursor cells (Pro-SG) into SSCs. To our knowledge, RHOX10 is the first factor shown to have a role in this developmental step. Given that Pro-SG are a transient and finite population of germ cells that are considered to be the soul source of SSCs, this is an important advance. Another goal of our study was to determine whether RHOXF2, a candidate human ortholog of mouse Rhox10, also functions in SSCs. Towards this goal, during the progress period, we optimized culture and lentivirus infection conditions for germ cells obtained from human testicular biopsies. We also initiated experiments to knockdown RHOXF2 in these cultures using RNA interference.