Decoding cytarabine-induced damage in the prepubertal testis: A multi-omics atlas of ecosystem and inheritance

Long-term survival after pediatric leukemia has improved, but chemotherapy-induced gonadotoxicity threatens future fertility, especially in prepubertal boys who cannot bank sperm. Using an integrated multi-omics framework in prepubertal mouse testes, we define how cytarabine (Ara-C) remodels the germ–somatic ecosystem. Ara-C induces dose-dependent architectural injury, endocrine suppression, and sperm loss. Single-cell analysis shows spermatogonia depletion via ferroptosis and selective arrest of DNA-replication programs in differentiating progenitors, while elongating spermatids activate maladaptive lipid-metabolic and endoplasmic-reticulum stress pathways. Sertoli cells shift from niche support to stress survival, marked by downregulated adhesion/trophic modules, a Klf9-driven antioxidant response, and TGFβ/BMP activation. Leydig cells reduce terminal steroidogenic enzymes and engage hypoxia- and inflammation-linked signaling, indicating endocrine impairment with paracrine immunomodulation. Cell–cell communication reveals a hyperconnected microenvironment with Sertoli cells as hubs, showing increased ACTIVIN, ADIPONECTIN, CSF, and GRN signaling and reduced PDGF, noncanonical WNT, and VEGF pathways. Unexposed F1 offspring of Ara-C–treated males retain inflammatory and metabolic alterations, indicating intergenerational imprinting. This atlas identifies actionable targets for fertility-preserving interventions.