Mitochondrial ROS-associated integrated stress response is involved in arsenic-induced blood-testis barrier disruption and protective effect of melatonin

• Post-weaning As exposure disrupts the BTB and reduces sperm counts in adult mice. • As decreases translational efficiency of barrier junction proteins in Sertoli cells. • As induces ISR by SIRT3 reduction-evoked mitochondrial dysfunction in Sertoli cells. • Melatonin reverses As-mediated mitochondrial dysfunction by SIRT3/ac-SOD2 axis. • Melatonin rescues As-mediated BTB disruption and sperm counts decline in adult mice. Arsenic (As) is an environmental metalloid. Previous studies have demonstrated that As exposure resulted in decline of sperm quality. This study aimed to investigate the impact of exposure to As on blood-testis barrier (BTB) in a mouse model. Four-week-old male mice were exposed to NaAsO 2 (1 or 15 mg/L) for 6 weeks. Our results found that NaAsO 2 exposure disrupted the BTB and reduced sperm counts in adult mice. NaAsO 2 activated the integrated stress response (ISR) and downregulated barrier junction protein in mouse testes and Sertoli cells. Ribosome profiling sequencing (Ribo-seq) and Ribosome-nascent chain complex-bound mRNA qPCR (RNC-qPCR) showed that translational efficiency of N-cadherin and ZO-1, two key barrier junction proteins, was reduced in NaAsO 2 -treated Sertoli cells. Mechanistically, NaAsO 2 exposure reduced SIRT3 protein via proteasomal degradation, thereby resulting in mitochondrial dysfunction and excess mitochondrial ROS (mtROS) generation in Sertoli cells. Melatonin alleviated NaAsO 2 -induced mitochondrial dysfunction and mtROS upregulation via reducing SOD2 acetylation in Sertoli cells. Moreover, melatonin antagonized NaAsO 2 -induced ISR, barrier junction proteins downregulation and barrier function impairment in Sertoli cells. Accordingly, melatonin attenuated NaAsO 2 -evoked BTB disruption and sperm count reduction in adult mice. These results suggest that mitochondrial dysfunction-associated translational inhibition of barrier junction proteins is involved in As-mediated BTB disruption and sperm quality decline.