A mouse model of busulfan-induced ovarian toxicity and evidence for attenuation with mTOR inhibition.

Busulfan, an alkylating agent used in hematopoietic stem cell transplantation conditioning, is associated with a high risk of ovarian toxicity. We established a time-resolved in vivo model of busulfan-induced ovarian injury and tested whether sapanisertib, an mTOR inhibitor, mitigates damage. Mice received busulfan (30 mg/kg, intraperitoneally) or saline; ovaries were assessed at 12, 24, and 72 h and 7 days by hematoxylin and eosin histology, follicle counting, TUNEL, and Ki-67 immunohistochemistry. A separate cohort received daily oral sapanisertib (0.3 mg/kg) from 7 days before busulfan through euthanasia; proliferation was assessed at 24 h, and follicle counts at 7 days. In the busulfan-only group, Ki-67 staining showed an early proliferative surge at 24 h in growing follicles, including primary follicles, whereas loss of primordial follicles and an increased primary-to-primordial follicle ratio were evident by 7 days. Primordial follicles showed no TUNEL-positive cells through 72 h, whereas growing follicles were frequently TUNEL-positive. Relative to busulfan alone, the mTOR inhibitor preserved primordial follicles, shifted the primary-to-primordial ratio toward control values, and reduced the proportion of primary follicles with extensive proliferation at 24 h. These findings are consistent with premature activation of primordial follicles as a proximate contributor to busulfan-induced loss of ovarian reserve and suggest that mTOR inhibition may mitigate this process. A two-time-point assay (24 h for proliferation, 7 days for morphology) provides a practical framework for future studies and translation.