High matrix stiffness triggers testosterone decline in aging males by disrupting stem Leydig cell pool homeostasis.

Aging is closely related to the decline of male reproductive endocrine function, which is manifested as insufficient testosterone production. It is well known that stem cell pool stability is crucial for maintaining tissue function. However, the relationship between aging and the stem Leydig cell (SLC) pool homeostasis remains unclear. Here, we demonstrate that extracellular matrix (ECM) stiffness increases in aging testes, and SLC pool homeostasis is imbalanced. Mechanistically, high ECM stiffness increases calcium influx mediated by Piezo1, leading to mitochondrial dysfunction and excessive reactive oxygen species (ROS). Excessive ROS promotes Gli1 degradation via the ubiquitin-proteasome pathway, ultimately inhibiting the proliferation and differentiation ability of SLCs. Together, these findings reveal the role of ECM stiffness, a biomechanical property in testes, in regulating SLC pool homeostasis and suggest that pretreatment of SLCs with low ECM stiffness in vitro may be an effective strategy for their expansion and for restoring testosterone levels in aging males.