Busulfan affects the transcript levels of mitochondrial dynamics regulators without cytotoxicity in rat Sertoli cells

Busulfan is a widely used alkylating chemotherapeutic agent known for its pronounced gonadotoxic effects, primarily attributed to the depletion of spermatogenic cells. However, the direct cellular responses of testicular somatic cells, particularly Sertoli cells (SCs), remain poorly understood. While busulfan has been reported to induce apoptosis in various proliferative cell types, accumulating in vivo evidence suggests that SCs exhibit resistance to chemotoxic injury. In this study, we investigated the direct effects of busulfan in vitro on freshly isolated primary SCs, with a particular focus on cell survival and mitochondrial dynamics, independent of germ-cell interactions. Our results demonstrate that busulfan does not induce apoptotic cell death in primary SCs, as evidenced by unchanged caspase activity and Annexin V staining following busulfan treatment. Instead, busulfan exposure leads to a distinct remodeling of mitochondrial dynamics. Key mitochondrial fission regulators, including Fis1 and Drp1, were significantly downregulated, indicating suppression of mitochondrial fragmentation. Concurrently, increased expression of the Mfn1 and Opa1 as key mediator of mitochondrial fusion was associated with reduced MTT assay readouts, reflecting a metabolically restrained state rather than cytotoxicity. This interpretation was further supported by decreased expression of P53 and other senescence-associated markers, suggesting attenuation of stress and DNA damage responses. Collectively, these findings indicate that SCs respond to busulfan exposure by adopting fusion-dominant, survival-oriented metabolic program that preserves mitochondrial integrity and cellular viability. This study provides direct evidence for the intrinsic resilience of SCs and highlights mitochondrial dynamics as a key adaptive mechanism underlying their resistance to busulfan-induced toxicity.