PRMT5 Inhibition Promotes Cross-Species Spermatogonia Expansion and Suppresses Differentiation

Spermatogonial stem cells (SSCs) hold great promise for treating male infertility, but their clinical translation is impeded by the lack of optimal conditions to maintain their undifferentiated state in vitro. In this study, we focused on epigenetic regulators upregulated during differentiation as potential targets. Through a small-molecule screen targeting such conserved regulators, we found that PRMT5 inhibition suppressed mouse SSC differentiation and enhanced their proliferation in a GDNF-deficient, differentiation-prone microenvironment in vitro. Using SSC transplantation assays, we confirmed that EPZ015666-treated SSCs retained their spermatogonial identity. This differentiation-inhibitory effect was reversible upon EPZ015666 withdrawal, allowing for the restoration of normal spermatogenesis. Notably, EPZ015666 also inhibited differentiation and promoted the proliferation of human and non-human primate spermatogonia in vitro. Mechanistically, EPZ015666 exerted this effect by inhibiting the enzymatic active site of PRMT5. These findings suggest that PRMT5 inhibition could provide a novel strategy for culturing human SSCs in vitro. Overall design: To address this, we generated Prmt5-knockout mouse cultured SSCs from Prmt5 flox/flox; Rosa26-CreERT2 mice by treating them with 1 µM 4-hydroxytamoxifen (4OHT) in vitro (Dong et al, 2021). Compared to the ethanol-treated (EtOH) control group, the expression level of PRMT5 and DMRT1 dramatically reduced in these knockout SSCs treated with 4OHT