PCB118暴露对青春期雄性ICR小鼠精子发生的影响及机制

Effect of PCB118 exposure on spermatogenesis in adolescent male ICR mice and mechanism

目的观察青春期雄性ICR小鼠暴露PCB118对精子发生的损害作用,探究其与睾酮合成过程相关的作用机制。方法36只4周龄雄性ICR小鼠随机分为3组,每组12只,每日ig给予PCB118(50和500μg·kg^(-1))或溶媒(玉米油),连续4周。给药结束后计算脏器系数;HE染色观察睾丸组织病理变化,统计曲细精管直径、生精上皮厚度和精原细胞相对数目;检测附睾精子数目、精子活力和精子畸形率;用放射免疫法检测血清睾酮水平;RT-qPCR法检测睾丸中与睾酮合成相关的酶和雄激素受体(Ar)mRNA水平;Western印迹法和免疫组织化学法检测AR蛋白和增殖细胞核抗原(PCNA)蛋白的表达。结果与溶媒对照组相比,PCB118500μg·kg^(-1)组肝系数明显下降(P<0.01),PCB1182个剂量组睾丸、附睾、肾和脾系数均无显著变化。PCB1182个剂量组睾丸组织中出现精原细胞缺失、曲细精管空泡化,曲细精管直径、生精上皮厚度和精原细胞相对数目均显著下降(P<0.05,P<0.01);附睾中精子数量显著减少,畸形率显著增加(P<0.05),但精子活力无显著变化;血清睾酮水平显著下降(P<0.05);睾丸中Ar及与睾酮合成相关的酶类固醇合成急性调节酶、胆固醇侧链裂解酶、3β-羟基类固醇脱氢酶、17-20裂解酶和17β-羟基类固醇脱氢酶的mRNA表达水平显著下降(P<0.05);AR和PCNA蛋白表达显著下降(P<0.05)。结论PCB118导致生精细胞增殖抑制,精子质量下降,该作用与其降低血清睾酮水平、下调睾酮合成相关酶mRNA水平、降低AR蛋白和mRNA表达以及降低PCNA蛋白表达有关。

OBJECTIVE To investigate the PCB118-induced damage to spermatogenesis in adoles⁃cent male ICR mice and the mechanism related to the process of testosterone synthesis.METHODS Thirty-six ICR male mice,aged 4 weeks,were randomly divided into 3 groups(12 mice per group)and treated with PCB118(50 and 500μg·kg^(-1),ig)and vehicle(corn oil)once daily for 4 weeks.The organ coefficient was calculated after the last treatment.The histopathological changes of testis tissues were detected by HE staining.The diameter of the seminiferous tubule,height of the spermatogenic epithelium and the relative number of spermatogonia were calculated.The quality of sperms,involving sperm count,sperm motility and total sperm deformity rate,was detected.The level of serum testosterone was assayed by radioimmunoassay,and the relative mRNA expressions of testosterone synthesisrelated enzymes and androgen receptor(Ar)in the testis were detected by RT-qPCR.The relative protein expressions of AR and proliferating cell nuclear antigen(PCNA)in the testis were analyzed by Western blotting and Immunohistochemistry.RESULTS Compared with the vehicle control group,there was no significant difference in the coefficients of the testis,epididymis,kidney and spleen in the two dose groups of PCB118,but the liver coefficient of the PCB118500μg·kg^(-1) group was reduced significantly(P<0.01).In the testis tissues of the two dose groups of PCB118,some spermatogonia were lost,and vacuoles appeared inside some seminiferous tubules.The diameter of the seminiferous tubule,height of the seminiferous epithelium and the relative number of spermatogonia decreased significantly(P<0.05,P<0.01).The number of sperms in the epididymis decreased significantly,while the rate of deformity increased significantly(P<0.05).However,there was no significant change in sperm motility.The serum testosterone level decreased significantly(P<0.05).The relative mRNA expressions of testosterone synthesis-related enzymes(steroidogenic acute regulatory,cholesterol side-chain cleavage enzyme,3beta-hydroxysteroid dehydrogenase,cytochrome P45017α-hydroxy⁃lase/17,20-lyase and 17beta-hydroxysteroid dehydrogenase)and Ar were decreased significantly(P<0.05),so were the relative protein expressions of PCNA and AR(P<0.05).CONCLUSION PCB118 can inhibit the proliferation of spermatogenic cells and reduce sperm production,which is related to lowering serum testosterone levels,down-regulating mRNA levels of testosterone synthesis-related enzymes,reducing protein and mRNA expressions of AR,and reducing protein expression of PCNA.