Functional robustness of adult spermatogonial stem cells after induction of hyperactive Hras.

Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of Hras(G12V), a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither Hras(G12V)-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous Hras(G12V) mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation. Author summary Recent research has found that advanced paternal age is associated with increased risk in children to develop a subset of congenital anomalies, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. The causative genetic errors (mutations) in these disorders have been identified to originate from the fathers' testicles and their numbers increase with fathers' age. It has been hypothesized that the germline stem cells that continuously self-renew and differentiate to supply sperm (referred as spermatogonial stem cells [SSCs]) carry these mutations and have the ability to expand preferentially as compared to normal SSCs with advancing age of the father, thereby increasing the likelihood of transmission of mutant sperm to the next generation. To test this hypothesis, we created a mouse model, in which a mutation known to enhance cell proliferation is induced in a subset of SSCs, and these cells compete with the neighboring normal (i.e., wild-type) stem cells. However, surprisingly, the germline cell population carrying the mutation in the testis was stable over a year of observation, suggesting that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.