Study Of Perinatal BDE-209 Exposure To Disrupt Blood-testis Barrier Of Mice Via Upgraduated ER? Signal Pathway

Objective:As an important environmental endocrine disruptors(EEDs),Decadrominated diphenyl ethers(BDE-209)is widely used in electronic devices,upholstery and carpets.BDE-209 has neurotoxicity and reproductive toxicity that interferes with the the synthesis,secretion,transport,binding,action,metabolism and elimination of hormones in vivo.Studies have shown that PBDEs concentration was negatively correlated with the newborn,reducing the weight of the epididymis,which exhibits early life toxicity.To explore the effect of testis estrogen receptor ?(ER?)exposure to BDE-209 on the blood testis of F1 male mice,ICR mice were used to investigate the effect of BDE-209 on the expression of testis ER?,Formin1,F-actin,blood-testis barrier(BTB)-related protein(claudin-11,occludin,ZO-1)and the disruption of BTB structure.In addition,SerW3 cell was co-treated with BDE-209 and Methyl-Piperidino-Pyrazole(MPP),the expression of ER?,Formin1 and other BTB related proteins were detected using Westerblot.This study provides a scientific basis for elucidating that BDE-209 exposure to disrupt blood-testis barrier of mice via upgraduated ER? signal pathway.Methods:In animal experiment,72 six-week-old mice(24 male and 48 female)was selected which feeded for 5 days adaptively.The mice were randomly divided into the control group(corn oil 100 mg / kg BW),BDE-209 100 mg / kg BW group,BDE-209 300 mg / kg BW group,BDE-209 500 mg / kg BW group.Pregnancy(GD)7 days after the F0 rats were orally administered BDE-209,treated once every morning,continuous feeding to postpartum(PND)21 days.Weighed and recorded the weight of rats(F0-PND21)every morning and the number of PND1 F1.In the PND35 and PND105 selected F1 male rats,remove the mouse eye to keep the blood sample,then cervical dislocation,separated organs in the ice bath,then stored organs in the corresponding conditions.The rest of the organs immediately placed in the liquid nitrogen tank frozen storage,and timely transfer to-80? refrigerator storage,when the need for biological samples to be detected,and prohibit repeated freezing and thawing.Real-time quantitative polymerase chain reaction(RT-PCR),immunoblotting,immunofluorescence chemiluminescence and immunohistochemistry were used to detected the expression of ER? mRNA and protein,BTB related proteins and Formin1 and F-actin proteins.In vitro,SerW3 cell was o-treated with different concentrations of BDE-209(0?g / ml,10?g / ml,50?g / ml,100?g / ml and 150?g / ml)and MPP.When the cell growth stable,the cell density was about 1 � 108.Cell viability was determined by MTT assay.The expressions of ER?,Formin1,F-actin and claudin-11,occludin and ZO-1 were detected by immunoblot and immunofluorescence.Results:(1)Peritoneal BDE-209 exposuredecreased F1 male mice body weight and changed organ coefficient: Compared with the control group,each BDE-209 group exist difference of the mean body weight of PND1-PND35 F1 mice(P<0.05).In the PND35 mice,the epididymis organs coefficient in the 100mg/kg BW group was lower than that in the other groups(P<0.05),in the PND105 mice,the testis coefficient in control group was higher than other BDE-209 groups(P<0.05).(2)Peritoneal BDE-209 exposure upregulated the expression of ER? mRNA and ER? in F1 male mice testis and MPP inhibited BDE-209 upregulated ER? of Ser W3 cells: In PND35,the expression of ER? mRNA in the 500mg/kg BW group was significantly higher than that in the control group(P<0.05).In PND105,the expression of ER? in the 300mg/kg BW group and the 500mg/kg BW group was significantly higher than that in the control group(P<0.05).The expression of ER? in the testes of F1 mice was observed by immunohistochemical method.The expression of ER? in the testes of PND35 was shown higher in 300mg/kg BW and 500mg/kg BW,in PND105,the expression of ER? was significantly increased in the 500 mg/kg BW group.The expression of ER? in BDE-209 group was significantly higher than that in control group.In cell experiments: the expression of ER? protein in SerW3 cells was significantly higher than that in control group(P<0.05).(3)Peritoneal BDE-209 exposure downregulated the expression of Formin1 in F1 male mice testis and MPP inhibited BDE-209 downregulated Formin1 of Ser W3 cells: In PND35,the expression of Formin1 in 300mg/kg BW and 500mg/kg BW group were significantly lower than that in control group(P<0.05),the expression of Formin1 in all BDE-209 group was significantly lower than that in control group(P<0.05)in PND105;In cell experiments: the expression of Formin1 in BDE-209 group of SerW3 cells was significantly lower than that in the control group(P<0.05).(4)Peritoneal BDE-209 exposure downregulated the expression of F-actin in F1 male mice testis and MPP inhibited BDE-209 downregulated F-actin of SerW3 cells: The expression of F-actin in the PND35,compare with control group,300mg/kg BW group,500 mg / kg BW group decreased(P<0.05),In cell experiments,the expression of F-actin in each BDE-209 group of SerW3 cells was significantly lower than that of the control group(P<0.05).(5)Peritoneal BDE-209 exposure downregulated the expression of BTB-related mRNA and proteins in F1 male mice testis and MPP inhibited BDE-209 downregulated BTB-related proteins of Ser W3 cells: In PND35 and PND105,The expression of claudin-11 mRNA in the 300 mg/kg BW and 500 mg / kg BW group were significantly lower than that in the control group(P<0.05).The BDE-209 groups expressed ZO-1 and occuldin mRNA were significantly lower than that in the control group(P<0.05).In PND35,the expression of claudin-11 in the testes showed different changes between 500mg/kg BW group and control group.In PND105,the 300mg/kg BW and 500mg/kg BW group expression of claudin-11 decreased compare with control group(P<0.05).In PND35 and PND105,the expression of ZO-1 in the 300 mg / kg BW group and the 500mg/kg BW group was lower than that in the control group(P<0.05).The expression of BTB-related protein in testis was observed by immunofluorescence.The expression of claudin-11 protein in PND35 and PND105 was also decreased in the 500mg/kg BW group,and the expression of claudin-11 in PND35 was obvious.The decrease of occludin PND35 and PND105 expression in each dose group showed a decrease in expression.ZO-1 showed only a decrease in the expression of ZO-1 during PND35,while the expression of ZO-1 in PND105 was lower and the expression of ZO-1 was not obvious.In cell experiments,the expression of claudin-11 protein and occludin protein in each BDE-209 group of SerW3 cells was lower than that of control(P<0.05).but the expression of ZO-1 protein in BDE-209 group showed no change compared with the control group.(6)Perinatal BDE-209 exposure induced F1 male mice histomorphological indicators: HE slices found two periods of mouse testicular tissue of the seminiferous tubules appear sperm damage and number of different degree of change,Microscopic electron microscopy in the dose group in the close connection structure of varying degrees of destruction,Sertoli cells within the organelles also varying degrees of change.Conclusion:BDE-209 can increase the expression of ER? in testis and SerW3 cells,and decrease the expression of Formin1,F-actin and BTB-related proteins,and damage the tight connection of the supportingcells in testicular tissue,suggesting that BDE-209 disrupts blood-testis barrier of mice via upgraduated ER? signal pathway.