The Role Of MCU-mediated Mitochondrial Calcium Overload In Cadmium-induced Apoptosis Of Mouse Spermatogonia

Objective Cadmium（Cd）is a toxic metal and also a well-known reproductive toxicant.Previous studies found that cadmium induced testicular spermatogonia apoptosis,and the molecular mechanism remains to be elucidated.This study mainly observed the effect of CdCl₂ on mitochondrial Ca²⁺homeostasis in mouse spermatogonia,and explored based on the IP3R-VDAC1-MCU signaling pathway,mitochondrial Ca²⁺overload in cadmium-induced spermatogonia apoptosis.Methods The mouse-derived spermatogonial cell line,GC-1 spg cells were divided into different groups:control,Cd,BA and BA+Cd groups.The cells in the control and BAPTA-AM groups were treated with PBS or 10μM BAPTA-AM individually.The cells in the Cd group were treated with CdCl₂（20μM）for 24h.The cells in the BA+Cd group were pretreated with BAPTA-AM（10μM）for 1h and then incubated with CdCl₂（20μM）for 24h.After CdCl₂ treatment for 24 hours,the cells were collected to detect the following indicators.The apoptosis was detected by flow cytometry with Annexin V-FITC/PI method.The ROS and the mitochondrial membrane potential（MMP）were detected using DCFH-DA and JC-1 as fluorescent probe,respectively.The cytoplasmic calcium concentrations were detected using Fluo-4 AM as a fluorescent probe.The mitochondrial calcium concentrations were detected using Rhod-2 AM as fluorescent probe.ER calcium concentrations were detected using Mag-Fluo-4 AM as fluorescent probe.RT-PCR was used to detect the m RNA expression levels of VDAC1、MCU、MCUR1.Western blot were used to detect the protein expression levels of Cyt c、AIF、p-IP3R/IP3R、VDAC、MCU、MCUR1.Results Compared with the control group,CdCl₂ treatment can cause intracellular reactive oxygen species to rise,mitochondrial membrane potential to decrease,and increased the apoptotic rate in spermatogonia.In addition,CdCl₂ exposure could induce the release of AIF and Cyt c from the mitochondria to the cytosol in spermatogonia.Further research found that CdCl₂ could activate IP3R-VDAC1-MCU pathways and cause mitochondrial Ca²⁺overload.BAPTA acetoxymethyl ester（BAPTA-AM）,a calcium chelator,almost completely attenuated IP3R phosphorylation,inhibited the m RNA and protein expression levels of VDAC1,MCU and MCUR1 upregulated by CdCl₂,reduced the calcium ion content in the mitochondria.Moreover,BAPTA-AM could decrease the level of ROS,antagonize CdCl₂-induced release of AIF and Cyt c from the mitochondria to the cytosol and alleviate apoptosis in spermatogonia induced by CdCl₂.Conclusion（1）CdCl₂ activated IP3R-VDAC1-MCU calcium regulation axis and caused mitochondrial Ca²⁺overload.（2）BAPTA-AM alleviated mitochondrial Ca²⁺overload and protected spermatogonia against CdCl₂-induced apoptosis via inhibiting IP3R-VDAC1-MCU signal pathway.（3）CdCl₂ exposure might induce apoptosis of GC-1 spg cells via mitochondrial Ca²⁺overload mediated by IP3R-VDAC1-MCU signal pathway.