高脂饮食诱导肥胖大鼠生精障碍及睾丸支持细胞线粒体和内质网结构功能的改变

Impairment of spermatogenesis and alterations in the structure and function of mitochondria and endoplasmic reticulum in Sertoli cells of high-fat diet-induced obese rats

目的观察高脂饮食诱导肥胖对大鼠生精功能的影响及睾丸支持细胞线粒体和内质网结构功能改变,探讨肥胖致生精障碍的可能机制。方法将32只雄性SD大鼠随机分为对照组和模型组,对照组给予普通饮食,模型组给予高脂饲料喂养。20周模型复制成功后解剖,取附睾进行精子功能分析;血清酶联免疫吸附试验分析性激素水平变化;苏木精-伊红染色观察肝脏和睾丸组织病理改变;油红O染色观察肝脏和睾丸组织脂质沉积情况;免疫荧光染色法检测睾丸闭锁小带蛋白1(ZO-1)、葡萄糖调节蛋白78(GRP78)及线粒体融合蛋白2(Mfn2)的定位及表达;Western blotting检测睾丸组织GRP78、Mfn2蛋白相对表达量;透射电镜观察睾丸支持细胞紧密连接及线粒体和内质网结构改变。结果模型组体重较对照组重(P<0.05),雌二醇较对照组高(P<0.05),睾酮较对照组低(P<0.05)。两组孕酮、泌乳素、促黄体生成素、卵泡刺激素比较,差异均无统计学意义(P>0.05)。模型组肝细胞脂肪变性明显,睾丸生精细胞排列稀疏,细胞变性,油红O染色均可见脂质沉积。对照组精子活力率高于模型组(P<0.05),畸形率低于模型组(P<0.05)。两组精子密度,直线速率和曲线速率比较,差异无统计学意义(P>0.05)。模型组GRP78蛋白相对表达量较对照组高(P<0.05),Mfn2蛋白相对表达量较对照组低(P<0.05)。模型组睾丸支持细胞间紧密连接分离,紧密连接蛋白ZO-1表达较对照组减少;内质网和线粒体肿胀,呈空泡状,部分网膜断裂。结论高脂饮食诱导肥胖可引起大鼠生精障碍,其机制可能与睾丸支持细胞线粒体和内质网结构功能改变导致紧密连接损伤有关。

Objective To observe the effects of obesity induced by high-fat diet on spermatogenic function and the structural and functional changes of mitochondria and endoplasmic reticulum in Sertoli cells of rats,and to explore the possible mechanism of impairment of spermatogenesis caused by obesity.Methods Thirty-two male SD rats were divided into the control group and the model group randomly.The control group was fed with normal diet,and the model group was fed with high-fat diet.After 20 weeks of modeling,the rats were sacrificed,and sperms in the epididymis were collected for the function analysis.The enzyme-linked immunosorbent assay was used to analyze the changes of sex hormone levels.The pathological changes of liver and testes were observed by hematoxylin-eosin staining.Lipid deposition in liver and testes was observed by oil red O staining.The localization and expressions of ZO-1,GRP78 and Mfn2 in the testes were detected by immunofluorescence staining.The relative protein expressions of GRP78 and Mfn-2 in testes were detected by Western blotting.Transmission electron microscopy(TEM)was used to observe the alterations in tight junctions between Sertoli cells and the structural changes of mitochondria and endoplasmic reticulum in Sertoli cells.Results The body weight in the model group was higher than that in the control group(P<0.05).The estradiol level in the model group was higher than that in the control group(P<0.05),while the testosterone level in the model group was lower than that in the control group(P<0.05).There was no difference in the levels of the progesterone,prolactin,luteinizing hormone and follicle stimulating hormone between the two groups(P>0.05).In the model group,the steatosis of hepatocytes was obvious,the arrangement of spermatogenic cells in the testes was sparse,cellular degeneration was observed,and lipid deposition was observed with oil red O staining.The sperm motility in the control group was higher than that in the model group(P<0.05),and the malformation rate of sperms in the control group was lower than that in the model group(P<0.05).There was no difference in the sperm density,straight-line velocity,and curvilinear velocity between the two groups(P>0.05).The relative protein expression of GRP78 in the model group was higher than that in the control group(P<0.05),whereas the relative protein expression of Mfn2 in the model group was lower than that in the control group(P<0.05).The tight junctions between Sertoli cells were disrupted in the model group,and the protein expression of ZO-1 was lower in the model group than in the control group.Besides,endoplasmic reticulum and mitochondria were swollen and vacuolar,and some of the membranes were ruptured in the model group.Conclusions Obesity induced by high-fat diet can cause impairment of spermatogenesis in rats,and its mechanism may be related to the damage of tight junctions caused by the structural and functional changes of mitochondria and endoplasmic reticulum in Sertoli cells.