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.
The goal of this Exploratory Concepts proposal is to test the hypothesis that a key regulatory factor, originally discovered by the applicant, plays a critical role in the biology of spermatogonial stem cells (SSCs), which are essential for the development of sperm. The biological functions and molecular targets of this gene will be investigated using a combination of molecular analyses and functional assays in both murine models (Aim 1) and human testicular-derived SSCs (Aim 2).
Novelty and Transformative Potential
- Reviewers agreed that human SSCs are relatively understudied compared to other stem cells and that the main hypothesis is novel. The gene under study has not been identified previously as a regulator of spermatogonial stem cell functions.
- The plan to extend discoveries from animal models into human cells is very significant.
- While the proposal does not clearly articulate how studies of the gene in question would address major gaps in our knowledge about the regulation of SSC self-renewal, most reviewers believed that a successful outcome could have a transformative impact on the field.
Feasibility and Experimental Design
- The applicant provides a wealth of preliminary data to support the rationale for the proposed studies.
- The use multiple of approaches for exploring SSC biology increases the likelihood of obtaining relevant insights. The use of xeno-transplantation for validation, in particular, was viewed as a major strength.
- Some reviewers questioned the feasibility of Aim 2, citing technical challenges associated with isolating, manipulating and culturing SSCs from human testes, as well as evaluating their functionality. There is scant evidence provided to support the applicant’s mastery of these methodologies, which will be critical for correct interpretation of results.
- The gene knockdown approaches proposed for Aim 2 may lead to a less penetrant phenotype. Reviewers suggested that TALEN or CRISPR technologies be considered for achieving a complete knock out in the human system.
Principal Investigator (PI) and Research Team
- The PI is a productive scientist with strong record of accomplishment in mouse genetics and transgenic biology. The PI’s lab has made seminal discoveries in the past.
- The team assembled has the expertise necessary to successfully conduct the proposed experiments.
Responsiveness to the RFA
- The proposed research is directly related to stem cell biology. The use of the mouse model system in Aim 1 is justified as necessary for translation to the human system.