| [Objective] In recent years,there have been continuous studies to prove that fluoride can damage the male reproductive system.However,its mechanism remains unclear.This experiment aims to elucidate the effect of fluoride on mitophagy in male mice testes,further explore its toxicity mechanism,and provide a new theoretical basis for explaining the male reproductive toxicity of fluoride.[Method] For in vivo experiment,40 male mature C57BL-6 mice(8 weeks)were randomly divided into 4 groups,10 mice in each group.The control group was treated with deionized water.The fluoride groups were treated with deionized water containing 25,50 and 100 mg/L Na F respectively.After 90 d,the mice were treated with 20 % Urethane solution,and sacrificed by neck amputation.Then,their testicular tissues were taken out quickly.The changes of mitochondrial ultrastructure and mitophagy of germ cells,Sertoli cells and Leydig cells were observed by transmission electron microscopy.And the expression and distribution of mitophagy receptor PHB2 in testicular tissue were detected by immunohistofluorescence.For in vitro experiment,TM3 Leydig cells were treated with 0,0.125,0.25,and 0.5 m M Na F for 24 h respectively.After that,the mitochondrial membrane potential changes were detected by JC-1 assay.And Mito Tracker Green(MTG)and Lyso Tracker Red(LTR)were used to trace mitochondria and lysosomes.Besides,the total m RNA and protein were extracted to detect the expression of genes and proteins related to mitophagy.[Results] 1.After 90 d of fluorine exposure,compared with the control group,more vacuoles,various degrees of cristae disorder and resolution and ruptured mitochondrial membrane were observed in germ cells,Sertoli cells and Leydig cells in testes of each treatment group.In addition,after 100 mg/L Na F treatment,typical mitophagosomes were observed in germ cells.So did the Leydig cells after treated with 50 and 100 mg/L Na F.However,typical mitophagosome was not observed in Sertoli cells.2.The results of immunohistofluorescence showed that the expression of PHB2 in testes increased significantly in the 50 and 100 mg/L Na F groups(p<0.01,p<0.01).In addition,PHB2 was mainly expressed in the Leydig cells.3.According to the results of MTT,IC50=2.939 m M after treatment of TM3 cells with Na F for 24 h,and IC50=1.992 m M after treatment of TM3 cells with Na F for 48 h.4.In the mitochondrial membrane potential assay by JC-1,after treated with 0.25 and 0.5 m M Na F for 24 h,the mitochondrial membrane potential of TM3 cells decreased significantly in a dose-dependent manner(p<0.01,p<0.01).However,there was no significant effect on the mitochondrial membrane potential of TM3 cells after 0.125 m M Na F exposure.5.After 24 h of fluoride exposure,MTG and LTR staining of TM3 cells showed that the number of mitochondria in the cells of each treatment group decreased significantly(p<0.01)and the number of lysosomes increased significantly(p<0.01),both in a dose-dependent manner compared with the control group.In addition,co-localization of MTG and LTR revealed that the number of lysosome-encapsulated mitochondria in each treatment group increased in a dose-dependent manner in comparison with the control group.6.The results of immunofluorescence showed that the expression levels of PINK1 and PHB2 were increased in the cells after fluorine treatment,and the number of mitochondria was decreased at the same time.The results of q RT-PCR and Western blot showed that the m RNA and protein expression levels of PINK1 were significantly increased in the 0.5 m M Na F treatment group(p<0.05),and there was no significant change in other treatment groups.And PHB2 m RNA expressions were significantly increased in all the treated groups(p<0.01).Meanwhile,its protein expression showed similar trends at 0.125,0.25 and 0.5 m M Na F and also increased significantly(p<0.05,p<0.01,p<0.05).[Conclusion] Fluoride could damage mitochondria in mouse testes and induce PINK1/Parkin pathway-mediated mitophagy in mouse Leydig cells and TM3 Leydig cells. |
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