Functional robustness of adult spermatogonial stem cells after induction of hyperactive <i>Hras</i>

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 HrasG12V, 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 HrasG12V-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 HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.越来越多的证据表明，父亲的年龄与子代的疾病风险存在关联。已知FGF-RAS-MAPK信号通路（FGF-RAS-MAPK signaling pathway）的从头功能获得性突变，会引发一类与父亲高龄相关的遗传病，例如阿佩尔综合征（Apert syndrome）、软骨发育不全（achondroplasia）、努南综合征（Noonan syndrome）以及科斯特洛综合征（Costello syndrome）。已有假说提出，携带致病性突变的成体精原干细胞会随时间发生克隆性扩增，并将突变传递给子代。然而，目前尚无能够在体内探究哺乳动物生殖系干细胞竞争行为的模型系统。本研究构建了一套谱系示踪系统，可使内源性表达HrasG12V的未分化精原细胞——该突变是RAS-MAPK信号通路中已确认的致病性功能获得性突变——在小鼠睾丸内与其野生型对应细胞发生竞争。经过长达一年的命运追踪分析，与野生型邻近细胞相比，HrasG12V阳性生殖细胞与精子均未出现显著的扩增现象。通过移植实验分析测得的短期干细胞能力，在野生型与突变组之间也无显著差异。此外，尽管在突变细胞系中可检测到组成型激活的HRAS蛋白，但这些细胞并未表现出增殖优势，也未对激动剂诱导的pERK信号通路产生增强应答。上述体内与体外实验结果表明，小鼠精原干细胞对内源性位点的杂合型HrasG12V突变具有功能抗性，且可能存在相关机制阻止此类有害突变发生扩增并传递给下一代。