Triptolide exposure triggers testicular vacuolization injury by disrupting the Sertoli cell junction and cytoskeletal organization via the AKT/mTOR signaling pathway

BACKGROUND: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure. METHODS: Male mice were subjected to TP at doses of 15, 30, and 60 μg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining. RESULTS: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells. CONCLUSION: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes.