| BackgroundDecabromodiphenyl ether(BDE-209),as a flame retardant,has been widely applied to various commercial products,as a kind of endocrine disruptor chemicals(EDCs),more and more evidence shows that spermatogenesis has a certain degree of male reproductive toxicity.Spermatogenesis goes through a series of complex changes,and spermiogenesis is the last stage of spermatogenesis,including nucleus enrichment and lengthening,acrosome formation,nucleoprotein transformation.Histone-to-protamine disorder affects the removal of excess chromatin in the process of sperm deformation and thus affects the plasticity of sperm head.Therefore,spermatogenesis plays an important role in sperm deformation.It is the spermiogenesis that needs to undergo complex changes,so it is extremely vulnerable to external factors.Autophagy is a unique protein degradation process.In recent years,there has been evidence that autophagy is related to sperm deformation.Previous studies have found that residual cytoplasts and sperm with round head in the epididymis of mice with decreased autophagy flux.However,whether BDE-209 interferes with spermatogenesis and spermatogenesis quality by changing autophagy flux and its potential mechanism remains unclear.ObjectiveIn this study,we used ICR male mice and GC-2spd cells as models to investigate the changes of autophagy pathway and the role and mechanism of histone to protamine conversion during the interference of BDE-209 in sperm deformation,which is of great significance to elucidate the mechanism of BDE-209 in the damage of sperm deformation.Methods1.In order to elucidate the effects of BDE-209 on reproductive injury,autophagy flux and sperm deformation in male mice,6-week-old ICR male mice were stably fed for one week.They were divided into corn oil control group and BDE-209 exposure group(exposure doses were 7.5 mg/kg BW,25mg/kg BW and 75 mg/kg BW)according to random number table method,with 12 animals in each group.Gavage was used for 42 days.Calculated and compared the organ coefficients in each group.HE staining,aniline blue staining and sperm immunofluorescence were used to observe the morphology of epididymal tissue and sperm morphology.The expression levels of autophagy related proteins P62 and LC3 B,transition proteins TNP1 and protamine PRM1 were detected by Western Blot and RT-PCR.2.In order to elucidate the mechanism of the effect of BDE-209 on autophagy flux,GC-2spd cells treated with 50μM BDE-209 were used as models to set DMSO,BDE-209 group,trehalose group,BDE-209+trehalose group,CQ group and BDE-209+CQ.To investigate whether BDE-209 inhibits the autophagy pathway of GC-2spd spermatocyte through AMPK-ULK1 pathway.DMSO,BDE-209 group,trehalose group and BDE-209+trehalose group,Compound C group and BDE-209+Compound C group were set.The expressions of autophagy related proteins P62 and LC3BII/I as well as pathway proteins AMPK and ULK1 were detected by Western Blot.3.In order to study the protective effect of trehalose on BDE-209 exposed mice,6-week-old ICR male mice were selected and fed under the same conditions,and divided into 4 groups according to the same method: corn oil control group,BDE-209 group(75 mg/kg BW),trehalose group and BDE-209+ trehalose group.Calculated and compared the organ coefficients in each group.HE staining,aniline blue staining and sperm immunofluorescence were used to observe the morphology of epididymal tissue and sperm morphology.the expression levels of autophagy related proteins P62 and LC3BII/I protein and m RNA were detected by Western Blot(Western Blot)and RT-PCR,and the changes of autophagosomes were observed by transmission electron microscopy.Western Blot(WB)and RT-PCR were used to detect the protein and m RNA expression levels of transition protein TNP1 and protamine PRM1.The chromatin removal in sperm head was observed by transmission electron microscopy.Results1.BDE-209 caused shrinkage of testis and epididymis,decreased coefficient of testis and epididymis organs,and decreased sperm quantity and quality.2.The effect of BDE-209 on sperm deformation.The residual excess chromatin and abnormal sperm head and flagella during sperm deformation suggest that BDE-209 may be affected during sperm deformation.Compared with corn oil control group,the expressions of transition protein TNP1 and protamine PRM1 decreased in BDE-209 groups,indicating histone to protamine conversion disorder(p < 0.05).3.Effect of BDE-209 on autophagy flux.In testis and GC-2spd cells,LC3BIII/I and P62 protein expression and m RNA expression were increased in BDE-209 groups compared with control group,suggesting that BDE-209 affects the changes of autophagy flux(p < 0.05)compared with autophagy flux inhibitor CQ group,LC3BII/I and P62 protein expressions increased in combined exposure group,and recovered in BDE-209+trehalose intervention group compared with trehalose intervention group.The results suggested that BDE-209 could affect the changes of autophagy flux,and trehalose could restore the changes of autophagy flux induced by BDE-209 exposure(p < 0.05).4.BDE-209 affected autophagy flux through AMPK-ULK1 pathway.In GC-2spd cells,compared with the control group,BDE-209 exposure increased pAMPK/AMPK and p-ULK1/ULK1 ratios.Intervention was conducted by AMPK specific inhibitor Compound C.We found that Compound C alleviated BDE-209 exposure and increased p-AMPK/AMPK and p-ULK1/ULK1 ratios.Meanwhile,trehalose intervention also alleviated BDE-209 exposure and increased pAMPK/AMPK and p-ULK1/ULK1 ratios.Thus,the expression of autophagy related proteins LC3BII/I and P62 in spermatocytes of GC-2spd cells was recovered.Therefore,through cell experiments,we found that trehalose alleviated the decrease of autophagy flux in spermatocytes of GC-2spd cells induced by BDE-209 exposure through AMPK-ULKI(p < 0.05).5.Abnormal expression of autophagy related protein and autophagosome in testis induced by BDE-209 exposure were restored by trehalose.In testis,compared with corn oil control group,the expressions of autophagy related proteins LC3BII/I and P62 were increased in BDE-209 group.Compared with the BDE-209 group,the expressions of autophagy related proteins LC3BII/I and P62 decreased in the BDE-209+ trehalose group.Transmission electron microscope observation showed that compared with BDE-209 group,BDE-209+trehalose group promoted the binding of autophagosomes and lysosomes,and recovered the number of autophagosomes,suggesting that trehalose could alleviate the decrease of autophagic flux and the number of autophagosomes in mouse testis caused by BDE-209 exposure.6.Trehalose restored BDE-209 on sperm deformation process.HE results showed that in the BDE-209+trehalose group,excess chromatin residue and sperm head and flagellum abnormalities were recovered during sperm deformation compared with the BDE-209 group.Compared with the BDE-209 group,the expressions of transition protein TNP1 and protamine PRM1 in the BDE-209+ trehalose group were recovered(p < 0.05),indicating that the histone protamine conversion disorder was recovered.The results suggested the influence of trehalose restored BDE-209 exposure on sperm deformation process.7.Trehalose restored the male reproductive toxicity induced by BDE-209,such as shrinkage of testis and epididymis,decreased coefficient of testis and epididymis organs,and decreased sperm quantity and quality.ConclusionsIn summary,this study found that:1.BDE-209 decreased autophagy flux and affected the conversion of histone to protamine and the process of spermiogenesis.2.BDE-209 induced sperm defects in mice by affecting autophagy flux through AMPK-ULK1 pathway.3.Trehalose restored autophagy flux and the histone to protamine conversion process caused by BDE-209 exposure,and the sperm deformation process was restored.4.Reduction of testicular organ coefficient,histomorphologic injury and sperm quality induced by trehalose restoration of BDE-209. |
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