ABSTRACT: Chemotherapy can compromise the fertility of boys with cancer, yet no standard protocols exist to preserve their reproductive potential. Before puberty, germ cells are almost exclusively spermatogonia that can be the target of anticancer drugs. Doxorubicin (DXO), a widely used anthracycline in pediatric oncology, has been associated with infertility in adulthood, but its immediate effects on prepubertal germ cells remain poorly understood. In the present study, a preclinical rat model of prepubertal DXO exposure was developed to characterize the mechanisms underlying immediate DXO-induced germ cell damage. Six-day-old pups, received a single intraperitoneal injection of DXO (5 mg/kg) and effects were measure after 24 or 48 h. DXO exposure significantly reduced relative testis weight from 24 h and significantly increased apoptosis and germ cell loss at 48 h, while circulating testosterone remained unchanged, suggesting a selective germline effect. RNA-seq was done on GFP-positive germ cells purified at 24 h. Transcriptomic analysis confirmed the enrichment in spermatogonial stem cells (SSCs) in the GFP-sorted population. Moreover, DXO induced 51 differentially expressed genes (49 upregulated, 2 down regulated) that were mostly related the p53-dependant apoptosis pathway. Pro-apoptotic genes (Cdkn1a, Bbc3/Puma, Tp53inp1, Fas) and oxidative stress regulators (Sesn2, Eda2r, Abhd4) were induced, whereas DNA repair genes (Mgmt, Xrcc1, Polh, Gadd45α, ...) were not activated. Our data revealed the DXO-induced immediate transcriptomic response after 24h, leading to germ cell death observed by histology at 48h. These findings suggest that SSCs respond to DXO by favoring apoptosis and stress regulation, a strategy that may preserve germline integrity and reduce the risk of transmitting genetic damage to the next generation.
