全氟辛酸诱导小鼠睾丸生精细胞凋亡过程中内质网应激途径的研究

Changes of Endoplasmic Reticulum Stress Signaling Pathway During Process of Apoptosis in Testicular Spermatogenic Cells of Mice Induced by Perfluorooctanoic Acid

全氟辛酸(perfluorootanoic acid, PFOA)是当前广受关注的环境污染物之一,通过饮食、饮水及粉尘摄入等途径进入机体,造成雄性生育能力下降。目前已有相关研究证实PFOA可诱导生精细胞凋亡,但是关于PFOA诱导生精细胞凋亡的机制研究还比较匮乏。因此本研究以雄性BALB/c小鼠为研究模型,基于内质网应激途径探讨PFOA诱导小鼠生精细胞凋亡的机制。将52只雄性BALB/c小鼠按随机数字表法随机分为4组:Control组、PFOA低、中和高剂量组(1.25、5和20 mg·kg^(-1)),PFOA处理组小鼠分别灌胃给予不同剂量的PFOA,Control组给予等体积的生理盐水,连续给药28 d后处死,取睾丸组织备用。苏木精-伊红(hematoxylin-eosin, HE)染色观察各组睾丸组织形态变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling, TUNEL染色)检测各组睾丸组织生精细胞凋亡,并计算凋亡指数;蛋白质印迹法(Western blot)检测Cleaved-Caspase-3、GRP78、CHOP、p-PERK、p-EIF2α、ATF4、p-IRE1、XBP1及ATF6表达水平变化;免疫荧光(immunofluorescence, IF)检测GRP78定位及表达水平。结果显示,PFOA可损伤小鼠睾丸生精结构,导致生精细胞脱落,此外PFOA还可诱导小鼠睾丸生精细胞凋亡,上调生精细胞凋亡指数,同时上调凋亡相关蛋白Cleaved-Caspase-3表达水平。随后Western blot结果显示PFOA可上调内质网应激标志蛋白GRP78及内质网应激介导的凋亡相关蛋白CHOP的表达水平,提示PFOA激活了内质网应激介导的凋亡信号通路。进一步研究发现,相比Control组,PFOA处理后睾丸组织内质网应激PERK信号通路相关蛋白p-PERK、p-EIF2α、ATF4上调,而内质网应激IRE1信号通路相关蛋白p-IRE1、XBP1和ATF6信号通路相关蛋白ATF6表达水平则无明显变化,提示PFOA暴露可特异性激活小鼠睾丸组织内质网应激PERK/EIF2α/ATF4信号通路。综上,PFOA可能通过激活内质网应激PERK/EIF2α/ATF4信号通路诱导生精细胞凋亡。

Perfluorootanoicacid(PFOA)is one of the environmental pollutants of widespread concern,which enters the host via diet,water and dust intake,finally resulting in decreased male fertility.Studies have found that PFOA can induce spermatogenic cell apoptosis,but the mechanism is yet unknown.Therefore,male BALB/c mice were used as the model in this study to explore the mechanism of PFOA-induced spermatogenic cell apoptosis via endo-plasmic reticulum stress pathway.Fifty-two BALB/c male mice aged 8 weeks were randomly divided into 4 groups:control group,three different doses of PFOA-treated groups.Mice in PFOA groups have been given different doses of PFOA(1.25,5 and 20 mg·kg^(-1))by oral gavage for 28 d,mice in control group were given equal volume of sa-line,and then all of them were sacrificed.Morphological changes of testis were observed by hematoxylin-eosin(HE)staining.The apoptosis of spermatogenic cells was detected by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL staining)and apoptosis index was calculated.The levels of Cleaved-Caspase-3,GRP78,CHOP,p-PERK,p-EIF2α,ATF4,p-IRE1,XBP1 and ATF6 were detected by Western blot.The localization and expression of GRR78 were detected by immunofluorescence(IF).Results showed that the normal structure of testicular tissues was damaged,and the number of shedding of spermatogenic cells was increased in PFOA-induced mice.In addition,PFOA induced the increase of apoptosis,up-regulation of the apoptosis index and increased expressions of apoptosis related protein Cleaved-Caspase-3 in spermatogenic cells.In addition,the ex-pression levels of endoplasmic reticulum stress marker protein GRP78 and endoplasmic reticulum stress-mediated apoptosis related protein CHOP were up-regulated in PFOA-induced mice,suggesting that PFOA activates endo-plasmic reticulum stress-mediated apoptosis signaling pathway.Furthermore,compared with the control group,PFOA caused the upregulation of PERK signaling pathway related proteins p-PERK,p-EIF2αand ATF4 in testis,however,there was no significant difference of the expression levels of p-IRE1,XBP1 and ATF6 after PFOA treat-ment,indicating that PFOA specifically activates the PERK/EIF2α/ATF4 signaling pathway in testis of mice.Taken together,PFOA may induce the apoptosis of spermatogenic cell by activating PERK/EIF2α/ATF4 signaling path-way.