TCDD宫内暴露致雄性子代大鼠生殖毒性研究

Reproductive toxicity of intrauterine exposure to 2，3，7，8-tetrachlorodibenzo-p-dioxin in male offspring rats

摘要:

目的：探讨2-3-7-8四氯二苯并二噁英（2-3-7-8 Tetrachlorodibenzo-p-Dioxin，TCDD）宫内暴露对雄性子代生殖毒性的影响及机制。方法：成功交配后，将母鼠随机分组。高剂量组（0.5 μg/kg）、低剂量组（0.1 μg/kg）和溶剂对照组，每组各6只，TCDD组及对照组于孕8~14 d（gestation day 8-14，GD8-14）采用灌胃方式分别给予TCDD及玉米油处理。孕鼠自然分娩，测量雄性仔鼠肛门至生殖器的距离（anal to genital distance，AGD）；计算睾丸的脏器系数；利用精子质量分析仪对仔鼠精子的质量及数量进行分析测定；酶联免疫吸附法对雄性子代血清睾酮（testosterone，T）水平进行测定；测定凋亡相关蛋白Bax、Bcl2、Caspase-3的表达水平。结果：0.1 μg/kg组、0.5 μg/kg组仔鼠与对照组仔鼠相比，AGD[（1.49±0.04） cm、（1.32±0.01） cm和（1.21±0.02） cm]有所缩短（P=0.000），精子数目[（72.33±4.46）×106个/mL、（63.67±3.44）×106个/mL、（45.33±5.47）×106个/mL]与精子活率[（78.53±1.26）%、（64.40±3.14）%、（53.87±3.65）%]明显降低，精子畸形率[（2.17±0.75）%、（4.83±0.75）%、（8.00±1.10）%]升高（P=0.000），血清中T水平[（2.68±0.06） ng/mL、（2.38±0.08） ng/mL和（2.10±0.09） ng/mL]明显下降（P=0.000），差异均有统计学意义，0.5 μg/kg TCDD组雄性仔鼠的睾丸质量（P=0.001）和睾丸脏器系数（P=0.004）降低的差异有统计学意义；睾丸组织细胞Bax蛋白相对表达量（0.836±0.004、1.185±0.004、1.414±0.001）随染毒剂量的递增而上升（P=0.000），Bcl-2（1.368±0.053、1.108±0.040、0.751±0.022）则相反（P=0.001），同时，0.5 μg/kg TCDD染毒组Pro-Caspase-3蛋白表达明显降低，但Cleaved-Caspase-3蛋白表达明显增加（P=0.000）。结论：宫内暴露TCDD可使雄性仔鼠出现雌性化特征，影响雄性仔鼠生育能力，对雄性子代具有生殖毒性，其机制可能与仔鼠睾丸组织细胞增殖-凋亡失衡和细胞凋亡的线粒体通路被激活有关。

Abstract:

Objective：To investigate the effect and mechanism of action of 2，3，7，8-tetrachlorodibenzo-p-dioxin（TCDD） intrauterine exposure on the reproductive function of male offspring rats. Methods：After mating，pregnant rats were randomly divided into high-dose TCDD group（0.5 μg/kg），low-dose TCDD group（0.1 μg/kg），and solvent control group，with 6 rats in each group. The TCDD groups and the control group were treated with TCDD and corn oil，respectively，by intragastric administration on gestation days 8-14 （GD8-14）. After being delivered naturally，male offspring were measured for anogenital distance（AGD），and testicular organ coeffi－cient was also calculated. Then the quality and quantity of sperm were analyzed by a sperm quality analyzer. In addition，the level of testosterone（T） in the serum of male offspring was determined by enzyme-linked immunosorbent assay，and the expression levels of the apoptosis-related proteins Bax，Bcl2，and caspase-3 were also measured. Results：Compared with the control group，both the low-dose group and high-dose group had a significantly shortened AGD[（1.32±0.01） cm and （1.21±0.02） cm vs. （1.49±0.04） cm，P=0.000]，and these two groups showed significant reductions in sperm count[（63.67±3.44）×106/mL and （45.33±5.47）×106/mL vs. （72.33±4.46）×106/mL] and sperm motility rate[（64.40±3.14）% and （53.87±3.65）% vs. （78.53±1.26）%]. Furthermore，a signifi－cantly increased rate of sperm deformity[（4.83±0.75）% and （8.00±1.10）% vs. （2.17±0.75）%，P=0.000] and a significantly decreased level of testosterone in serum[（2.38±0.08） ng/mL and （2.10±0.09） ng/mL vs. （2.68±0.06） ng/mL，P=0.000] were also observed in these two groups. The testicular weight and testicular organ coefficient in the high-dose group decreased significantly compared with those in the low-dose group（P=0.001 and 0.004，respectively）. With an increase in TCDD dose，the relative expression level of Bax protein increased significantly in testic－ular tissue cells（0.836±0.004 vs. 1.185±0.004 vs. 1.414±0.001，P=0.000），whereas the expression level of Bcl-2 demonstrated an opposite trend（1.368±0.053 vs. 1.108±0.040 vs. 0.751±0.022，P=0.001）. Meanwhile，the high-dose group had a significant reduc－tion in the expression level of pro-caspase-3 and a significant increase in the expression level of cleaved-caspase-3（P=0.000）. Conclusion：Intrauterine exposure to TCDD can induce feminization of male offspring，affect their fertility，and cause reproductive toxicity. The mechanism may be related to cell proliferation-apoptosis imbalance and activation of the mitochondrial pathway of apop－tosis in the testicular tissue cells of these offsprings.