Study On The Role Of Estrogen Receptor ? In Damage On Spermatogonia Induced By BDE-209

Objective Decabromodiphenyl ether(BDE-209)is a homologue of polybrominated diphenyl ethers(PBDEs).It is added to daily products as a flame retardant and can accumulate in human adipose tissue as an endocrine disruptor.However,little is known about the mechanism of BDE-209 exposure on testicular spermatogenesis.The purpose of this study was to investigate the possible mechanism of estrogen receptor alpha(ER?)in testicular reproductive disorders caused by BDE-209 exposure,and to understand the types of damaged cells and the stages of spermatogenic disorders caused by BDE-209.Methods Twenty-four male SD rats were randomly divided into DMSO solvent 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.A BDE-209 exposure on Male SD rat model via testis was established to collecting biological materials after a spermatogenic cycle.Weigh the testis,epididymis and gonads to calculate the organ coefficients.Analyze the chromatin concentration of sperm with a transmission electron microscope.Observe the general changes in sperm morphology with a sperm smear.The testicles Paraffin sections were stained with HE and PAS to observe testicular morphological changes.The expression of ER?,PLZF,c Kit,Sox9,Wnt3 a and Wnt5 a in testis of each group were detected by immunofluorescence.The expression of ER?,PLZF,c Kit,Sox9,Wnt3 a,Wnt5a,BRDT and GSK-3? in testes of each group were detected by Western Blot(WB).Another 24 male adult SD rats were randomly divided into DMSO group,BDE-209 group,4,4',4''-(4-propyl-[1h]-pyrazole-1,3,5-triyl)trisphenol(PPT)group and BDE-209+PPT combination group.A BDE-209 exposure via testis on male SD rat model was established.Mixture of BDE-209 and PPT was injected into testis in BDE-209+PPT combination group.Collect biological materials after a spermatogenic cycle in rats to weigh the testes,epididymis and gonads,then calculate the organ coefficients.Aniline blue staining was applied to observe sperm chromatin damage in epididymis tail.Paraffin sections were stained with HE and PAS to observe the morphological changes of testis in each group.Immunofluorescence was applied to detect the expression of ER?,PLZF,c Kit,Sox9,Wnt3 a and Wnt5 a in each group.WB was used to detect the expression of ER?,PLZF,c Kit,Sox9,Wnt3 a,Wnt5a,BRDT and GSK3-? in testis of each group.q RT-PCR was applied to detect the m RNA expression of ER?,PLZF,c Kit,Sox9,Wnt3 a and Wnt5 a in testis of each group.Results(1)BDE-209 caused reproductive toxicity in SD male rats.Compared with the DMSO solvent control group,the testis of each BDE-209 group showed atrophy to varying degrees.Compared with the DMSO solvent control group,testicular coefficient and sperm count decreased in different BDE-209 group.Compared with the DMSO solvent control group,chromatin concentration was impaired in BDE-209 group.The result of HE and PAS staining revealed damage of seminiferous tubules to different degrees,and immature spermatogenic cells shed in the lumen,indicating that the spermatogonia was severely damaged.Injury of spermatogonia tends to worsen with increasing BDE-209 dose.(2)BDE-209 down-regulated the expression of ER? in the testis of SD male rats,causing morphological damage to spermatogonia and reducing expression of marker protein in spermatogonia.The results of WB showed that compared with the DMSO solvent control group,the expression of testicular ER? decreased in each BDE-209 group(P<0.05).The results of Immunofluorescence are consistent with WB.Spermatogonia were damaged in the seminiferous tubules instead of sertoli cells.WB was applied to detect the expression of spermatogonia-related markers,namely PLZF and c Kit.The results of WB revealed that compared with the DMSO solvent control group,the level of PLZF expression was reduced in each BDE-209 group(P<0.05).The level of c Kit expression was not significantly different between groups.The expression of Sox9 was increased in 2.5 mg/kg BW BDE-209 group(P<0.05).The results of Immunofluorescence showed that compared with the DMSO solvent control group,the expression of PLZF and c Kit in each BDE-209 group was significantly reduced,while the expression of Sox9 was slightly increased.(3)Exposure of BDE-209 caused changes of Wnt signal in the testis.The data of WB showed that the protein expression of Wnt3 a in 0.1 mg/kg and 0.5 mg/kg BW BDE-209 group was higher than that in DMSO solvent control group(P<0.05),and the expression level of in 0.5 mg/kg and 2.5 mg/kg BW BDE-209 group was increase compared to DMSO solvent control group(P<0.05).GSK3-? in the testis of 2.5mg/kg BW BDE-209 group was increased(P<0.05).The result of Immunofluorescence showed that compared with DMSO solvent control group,the expression of Wnt3 a and Wnt5 a in 0.5 mg/kg BW BDE-209 group was significantly increased,and the expression of Wnt3 a in the 2.5 mg/kg BW BDE-209 group was significantly increased.(4)PPT up-regulated ER? in the testis of SD male mice,alleviated spermatogonia damage caused by BDE-209,reduced the degree of testicular coefficient decline,sperm count decline and chromatin concentration damage.After co-treatment with PPT and BDE-209,the protein and m RNA levels of ER? in the testes of rats increased significantly(P<0.05),and the tissue structure of seminiferous tubules in rat recovered.No obvious spermatogenic cell necrosis was seen in HE and PAS staining.Compared with the BDE-209 group,the expression of PLZF and c Kit in the BDE-209+PPT combination group increased(P<0.05).The result of quantitative real-time Polymerase Chain Reaction(q RT-PCR)found compared with BDE-209 group,the m RNA expression of PLZF was significantly increased(P<0.05).(5)Changes of Wnt signal in testis are suppressed after up-regulating ER?.Compared with the BDE-209 group,the protein and m RNA levels of Wnt3 a and Wnt5 a in the testis of BDE-209+PPT combination group were reduced to varying degrees(P<0.05).Conclusions(1)BDE-209 caused down-regulation of ER? and change of Wnt signal in testis of SD male rat,leading to reproductive toxicity and damage to spermatogonia during spermatogenesis.(2)After co-treatment with ER? agonist PPT and BDE-209,down-regulation of ER? and changes of Wnt signal in SD male rats' testis were alleviated,the reproductive toxicity effect caused by BDE-209 was restored,and the degree of spermatogonia damage was reduced.