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.
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.