| Objective Decabromodiphenyl ether(BDE-209)is an environmental pollutant with persistent and bioaccumulative properties that has been detected in the environment,wildlife and humans around the world in recent years.BDE-209 mainly causes damage to nerve and nerve development,immunity,embryo,reproduction,teratogenicity and(potential)carcinogenicity,among which male reproductive toxicity is receiving increasing attention.BDE-209 can disrupt the blood-testis barrier(BTB)structure during spermatogenesis,but the mechanism remains unclear.In this study,we have explored the important role of estrogen receptor alpha(ERα)on the destruction of BTB structure by BDE-209 in vivo and in vitro.Methods 60-day-old SPF Sprague-Dawley male rats were randomly divided into four groups:ehicle control group,0.1 mg/kg BW BDE-209 group,0.5 mg/kg BW BDE-209 group and 2.5 mg/kg BW BDE-209 group.Rats were exposed to testicular injection and sacrificed by cervical dislocation after 12.8 days.Sertoli cells were isolated from testes of 18-20 day old Sprague-Dawley male pups.Treatment of cells with BDE-209 after 72 h of culture,the doses were 12.5,25,50 μM,and the exposure time was 6 h,12 h,24 h,36 h,48 h,respectively.Screen the most appropriate dose and time for PPT treatment.There were five treatment groups: DMSO group,PPT group,BDE-209 group,BDE-209+PPT group and MPP group,and the exposure time was 24 h.Carry out the following experiments:(1)To observe the toxic effects of BDE-209 on SD male rats and primary Sertoli cells,the testicular organ coefficient of rats were calculated after BDE-209 infected.HE staining and PAS staining were used to observe the morphological damage of the testes and the seminiferous tubules of SD male rats after BDE-209 testicular injection.The MTT assay were used to detect the viability of cells in different concentrations.(2)To observe the effect of BDE-209 on the expression of ERα in SD male rats and primary Sertoli cells,the expression of ERα mRNA and protein were detected by qRT-PCR and WB assays.(3)To observe the structural and functional changes of BTB in SD male rats and primary Sertoli cells induced by BDE-209,we used ultrastructural electron microscopy to observe the BTB structure of rat testes.The mRNA,protein and positional expression of BTB-related proteins were detected by qRT-PCR,WB and immunofluorescence staining.(4)To elucidate the role of ERα in the destruction of BTB structure in primary Sertoli cells by BDE-209,qRT-PCR,WB and immunofluorescence staining were used to detect the mRNA,protein and positional expression of BTB-related proteins after co-treatment of ERα-specific agonist PPT and BDE-209.Results(1)Toxic effects of BDE-209 on SD male rats and primary Sertoli cells.Compared with the control group,the testicular weight of each dose group decreased(P < 0.05),and the testicular coefficient of 0.5,2.5 mg/kg BW group decreased(P < 0.05).HE staining were performed to observe eosinophilic granules and immature germ cells in the lumen of each dose group,sertoli cells vacuolation,long sperm insertion into the endothelium,interstitial inflammatory cell infiltration and the number of spermatogenic cells at all levels reduced or absent.PAS staining showed that the above lesions appeared in theseminiferous tubules of each stage of I-XIV phase.As the concentration and time of exposure increased,the viability of Sertoli cells gradually decreased.There was a statistically significant difference when the concentration of BDE-209 exceeded 50 μM compared with the control group(P < 0.05).(2)Effect of BDE-209 on ERα in SD male rats and primary Sertoli cells.Compared with the control group,the expression of ERα mRNA and protein were decreased in 2.5mg/kg BW group(P < 0.05).Compared with the control group,the expression of ERαprotein was decreased in primary Sertoli cells at 50 μM and 24 h,36 h,48 h(P < 0.05).(3)Effects of BDE-209 on the structure and function of BTB in SD male rats and primary Sertoli cells.2.5 mg/kg BW dose group showed obvious breakage or even disintegration in tight junction,loss of actin microfilaments,lipid droplets and a large number of vacuoles appear in the Sertoli cells.In animal experiments,compared with the control group,the mRNA expression of ZO1,Occludin,β-catenin and Formin1 decreased in each dose group(P < 0.05).The mRNA expression of N-cadherin and Eps8 decreased in the 2.5 mg/kg BW group(P < 0.05).Compared with the control group,the protein expression of ZO1,Occludin,Eps8 and Formin1 decreased in the exposed group(P < 0.05).In primary Sertoli cells,compared with the control group,the mRNA expression of Occludin and β-catenin decreased(P < 0.05).Compared with the control group,the protein expression of ZO1,Occludin,Eps8 and Formin1 decreased in the exposed group(P < 0.05),the protein expression of ZO1,Occludin,Eps8 and Formin1 decreased at 24 h,36 h,48 h(P < 0.05).Immunofluorescence experiments showed that after 12 hours of exposure,F-actin was sparsely arranged and truncated or arranged disordered.In the 2.5 mg/kg BW group,the expression of BTB-related protein was weakened,and it was tightly located around the nucleus and no longer localized on the cell surface.(4)PPT attenuated the effects of BDE-209 on ERα expression and BTB structure in primary Sertoli cells.The mRNA expression of Occludin and β-catenin decreased after BDE-209 treatment(P < 0.05),and the mRNA expression of Occludin and β-catenin in PPT and BDE-209 co-treatment group increased compared with BDE-209 group(P <0.05).Compared with the BDE-209 group,the protein expression of ZO1,Occludin,Eps8 and Formin1 were up-regulated in the co-treatment group(P < 0.05),the expression of BTB-related protein in the co-treatment group showed a recovery trend,and Formin1 showed a neatly arranged microfilament bundle.Conclusions BDE-209 exposure caused down-regulation of ERα expression and destruction of BTB structure in SD male rats testis and primary sertoli cells,and caused testicular morphology damage.PPT treatment can restore the down-regulation of ERα and BTB-related proteins after BDE-209 exposure,suggesting that estrogen receptor alpha mediates BTB-induced structural damage caused by BDE-209.Learn more about the molecular mechanisms by which BDE-209 causes spermatogenesis disorders and induces male reproductive damage.Looking for sensitive effect markers for screening male reproductive damage,providing a scientific basis for the prevention of male reproductive function damage of BDE-209 environmental endocrine disruptors. |
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