从内质网应激研究高脂饮食对小鼠睾丸生精细胞凋亡的影响

Effects of high-fat diet on testicular germ cell apoptosis in mice through endoplasmic reticulum stress

目的从内质网应激的角度研究高脂饮食对小鼠睾丸生精细胞凋亡的影响。方法将C57BL/6J雄性小鼠随机分为正常组和高脂饮食组,每组6只,分别给予普通饲料和高脂饲料持续喂养5个月。小鼠称体质量,麻醉后处死,取睾丸组织,称质量并计算睾丸指数;取附睾组织进行精液分析;HE染色检测睾丸组织形态学变化;TUNEL染色检测生精细胞凋亡;Western blot检测睾丸凋亡及内质网应激相关蛋白的表达水平;免疫荧光法检测GRP78蛋白表达和定位的变化。结果与正常组相比,高脂饮食组小鼠体质量明显增加,睾丸指数明显下降;精子浓度和精子活率明显下降;睾丸生精细胞排列松散,生精上皮厚度明显变薄,生精小管直径无明显变化;生精细胞凋亡增多,凋亡指数明显增加;Bax和cleaved-caspase-12蛋白的表达明显增加,Bcl-2蛋白表达明显降低;GRP78,p-IRE1,XBP1,ATF6α蛋白的表达水平明显上调,而p-PERK,p-eIF2α,ATF4蛋白的表达无明显变化,免疫荧光结果进一步显示睾丸组织中GRP78的表达明显增加,表达位置无明显变化。结论高脂饮食可诱导小鼠睾丸生精细胞凋亡,其机制可能与激活内质网应激IRE1信号通路和ATF6信号通路有关。

Aim To study the effects of high-fat diet on testicular germ cell apoptosis in mice through endoplasmic reticulum stress.Methods C57BL/6J male mice were assigned into normal group and high-fat diet group randomly,with six mice in each group.The mice in normal group or high-fat diet group were fed with regular or high-fat diet continuously for five months.The mice were weighed,anesthetized,and euthanized to collect testicular and epididymal tissue for analysis.The testicular tissue was weighed and their indices were calculated.Epididymal tissue was collected for semen analysis.The morphological alterations of testicular tissue were observed using hematoxylin-eosin(HE)staining.The apoptosis of germ cells was detected by TUNEL staining and the apoptotic indices were calculated.The expression levels of apoptosis and endoplasmic reticulum stress-related proteins in testicular tissue were detected by Western blot.The protein expression and localization of GRP78 in testicular tissue were further detected by immunofluorescence.Results The results showed that compared to the normal group,the high-fat diet group had a significant increase in body weight,a significant decrease in testicular index,sperm concentration,and sperm vability,loose arrangement of germ cells,significant thinning of the seminiferous epithelium,no significant change in the diameter of seminiferous tubules,a significant increase in germ cell apoptosis,with an increased apoptosis index,and significant increase in expression of Bax and cleaved-caspase-12,and a significant decrease in Bcl-2 protein expression.The expression levels of GRP78,p-IRE1,XBP1,and ATF6αproteins were significantly up-regulated,while p-PERK,p-eIF2α,ATF4 protein expression showed no significant changes.Immunofluorescence results further showed a significant increase in the expression of GRP78 protein in the testicular tissue,with no significant changes in the expression location.Conclusions High-fat diet can induce the apoptosis of mouse testicular germ cells,and the mechanism may be related to the activation of endoplasmic reticulum stress IRE1 and ATF6 signaling pathway.