六氯联苯诱导新生雄性大鼠睾丸细胞凋亡的体内体外研究
Hexachlorobiphenyl-induced Apoptosis in Germ Cells and Sertoli Cells of Neonatal Male Rats in Vivo and in Vitro
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摘要:
目的 观察2, 2', 4, 4', 5, 5'-六氯联苯(PCB153)对新生SD大鼠睾丸细胞凋亡的影响。 方法 体内实验:将出生3 d(postnatal day 3, PND 3)的SD大鼠随机分组,每组24只,经口给予溶剂对照(玉米油)和PCB153(0.025、0.250、2.500 mg/kg),连续暴露5 d。最后一次暴露24 h后解剖每组一半的动物,取睾丸组织做成切片,原位末端凋亡法(TUNEL法)检测睾丸细胞凋亡水平并电镜观察细胞形态;剩余动物在无暴露条件下继续喂养至12周龄(成鼠)解剖,进行精子计数。体外实验:新生大鼠(postnatal day 5, PND5)睾丸组织分离精原-支持细胞共培养48 h后,用0、1、10、50μmol/L的PCB153染毒24 h,染色观察细胞核凋亡,流式细胞仪测定细胞早期凋亡率和晚期凋亡率。 结果 新生期大鼠不同剂量PCB153暴露后,与对照组相比, 0.250 mg/kg和2.500 mg/kg剂量组12周龄睾丸每日精子生成量明显下降(P<0.05)。PND8时, TUNEL法检测发现PCB153染毒组大鼠睾丸中凋亡细胞各组间差异无统计学意义。电镜下观察曲细精管,可见染毒组支持细胞线粒体肿胀,生殖细胞核固缩凝结。体外共培养细胞染毒24 h后,流式细胞仪检测发现,与对照组比较, 50μmol/L组早期细胞凋亡率明显升高(P<0.05);但晚期凋亡率各组差异无统计学意义。荧光显微镜下观察,从PCB153染毒浓度10μmol/L起可见明显的凋亡细胞核形态特征。 结论 新生期大鼠暴露PCB153可直接诱导睾丸细胞凋亡,引起雄性大鼠生殖功能的远期损害。 Abstract:Objective To investigate the effects of neonatal exposure of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (PCB153) on apoptosis both in vivo and in vitro in male SD rats. Methods In vivo experiments:neonatal SD rats (postnatal day 3, PND 3) were randomly divided into 4 groups and received oral administrations of PCB153 (0.025, 0.250, 2.500 mg/kg)or vehicle control for 5 d. Half of the rats were killed in 24 h after the final administration. The remains were fed until 12 weeks. The histological changes of rat testicular tissue of PND8 rats were examined by electronic microscope. The apoptosis and count of germ cells were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL). The daily sperm production of 12 week adult rats was recorded. In vitro experiments:PCB153 (0, 1, 10 and 50 μmol/L) were exposed to co-culture system for 24 h after germ cells and sertoli cells were co-cultured for 48 h. AnnexinV-fluorescein isothiocyanat (FITC) and propidium iodide was used for detecting early and late apoptosis rate by flow cytometer (FCM); 4', 6-diamidino-2-phenylindole (DAPI)staining was for observing apoptosis in cell nuclei. Results The daily sperm product in the 0.250 mg/kg and the 2.500 mg/kg dose groups were significantly reduced compared with that of the contro(l P < 0.05). PCB153 was not found to induce apoptosis in the germ cells by TUNEL detection. Electron microscopy revealed mitochondria swelled in the sertoli cells and nucleus pyknosis in the germ cells after 24 h PCB153-treated in vivo. In the 50 μmol/L group, the early apoptosis rate was significantly higher than the control group tested by FCM (P < 0.05). Apoptotic cell nuclei in the 10 μmol/L group were significantly increased compared with the control group. Conclusion Neonatal rats exposed to PCB153 could induce apoptosis both in vivo and in vitro at certain dosage. The lo ng-term damage to male reproductive function is possibly caused by neonatal exposure to chemicals.
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