Effect Of Mitochondria-associated Membrane Related Mitochondrial Calcium Overload On DEHP-induced Prepubertal Testicular Injury

Objective: To confirm the reproductive toxicity of diethylhexyl phthalate(DEHP)on prepubertal male mice,and to elucidate the underlying mechanisms of DEHP or MEHP(the principle metabolite of DEHP)induced prepubertal testicular injury.Methods: Part I: Male C57/BL6 J mice were daily intragastric administrated with 0,100,250,500 mg/kg·day of DEHP from postnatal day22(PND22)to PND35.(1)The testicular coefficient,histopathology and serum testosterone level of each mice were detected to confirm the reproductive toxicity of DEHP on male prepubertal mice.(2)Transcriptome sequencing and bioinformatic analysis combined with experimental verification were conducted to explore the potential mechanisms.Part ?: Cell lines of normal Leydig(TM3)and Sertoli(TM4)cells were cultured and administrated with different concentrations of MEHP.(1)Cell viability and apoptosis were detected to evaluate the toxicity of MEHP on Leydig and Sertoli cells.(2)Transcriptome sequencing and bioinformatic analysis were performed to explore potential mechanisms underlying MEHP induced Leydig and Sertoli cell injury.(3)Reactive oxygen species(ROS)level and oxidative stress damage-related enzymes were detected.Further,cells were exposed to MEHP combined with an ROS scavenger N-Acetyl-L-cysteine(NAC),and cell viability and apoptosis were assessed to confirm the involvement of ROS in MEHP induced Leydig and Sertoli cell damage.(4)Mitochondrial injury,mitochondrial membrane potential(MMP)and mitochondrial morphology were analyzed to evaluate the mitochondrial damage of Leydig and Sertoli cells(5)Changes of mitochondrial calcium level,mitochondria-associated membranes(MAMs)and the IP3R3-Grp75-VDAC1-MCU complex were detected.Moreover,TM3 and TM4 cells were co-treated with MEHP and the MCU inhibitor Ru360 to confirm the involvement of MAMs-related mitochondrial calcium overload in MEHP induced apoptosis by detection of cell viability and apoptosis rates.Results: Part I: Prepubertal exposure of DEHP may lead to testicular injury,including decrease of testicular coefficient,disturbance of seminiferous tubules and decline of serum testosterone level.Transcriptomic and bioinformatic analysis reveal that DEHP induces oxidative damage and cell apoptosis of testicular tissues,furthermore,it suggests that DEHP may damage the Leydig and Sertoli cells of prepubertal testes.Part ?: MEHP decreases cell viability and increases apoptosis rate of Leydig and Sertoli cells in vitro.Transcriptomic analysis and further experimental verification revealed that:(1)MEHP may induce intrinsic apoptosis,ROS overload and oxidative stress injury in Leydig and Sertoli cells.Co-treatment of NAC with MEHP may relieve the damages caused by MEHP exposure.(2)MEHP induces mitochondrial damage,promotes mitochondria fission and decreases MMP in Leydig and Sertoli cells.(3)MEHP induces mitochondrial calcium overload of Leydig and Sertoli cells.(4)MEHP increases the formation of MAMs,upregulates the protein level of IP3R3,Grp75,VDAC1 and MCU,and enhances the interaction between Grp75 and VDAC1 as well as Grp75 and IP3R3.Co-treatment of Ru360 may reduce the MEHP induced damage of Leydig and Sertoli cells.Conclusions: Prepubertal exposure of DEHP may induce testicular injury in mice.Cell apoptosis and mitochondrial damage induced by MAMs-mediated mitochondrial calcium overload are involved in MEHP induced Leydig and Sertoli cell iinjury.