妊娠期砷暴露对F1代成年雄性大鼠睾酮合成和精子质量的影响

Impact of arsenic exposure during gestation on testosterone synthesis and sperm quality in F1 adult male rats

  • 摘要:
    背景 砷是一种发育毒物,可通过胎盘屏障影响子代健康,但妊娠期环境水平砷暴露对子代成年雄性生殖功能的影响有待探究。
    目的 探讨妊娠期环境水平砷暴露对F1代成年雄鼠睾酮合成及精子质量的影响。
    方法 40只性成熟Wistar雌鼠按体重随机分为4组,分别为对照组和低、中、高剂量亚砷酸钠(NaAsO2)暴露组,每组10只,经适应性喂养后与性成熟Wistar雄鼠按2∶1进行合笼,以发现阴栓或阴道涂片观察到精子日确认为受孕日(GD0)。孕鼠从GD0起开始干预直至分娩出F1代,低、中、高剂量组分别摄入含1、5 、25 mg·L−1 NaASO2的饮用水,对照组给予不含砷的双蒸水。F1代雄鼠于70日龄处死,测量肛殖距离,取睾丸、附睾称重并计算脏器系数;测定附睾精子质量;观察睾丸组织学变化;利用ELISA试剂盒测定血清睾酮及雌二醇水平;采用实时荧光定量PCR技术测定睾丸类固醇激素关键酶的mRNA相对表达水平;采用Western blotting测定睾丸类固醇激素合成关键酶的蛋白相对表达水平。
    结果 与对照组相比,低、中剂量组的睾丸及附睾脏器系数增大(P<0.05),高剂量组的附睾脏器系数也增大(P<0.05)。在精子活动度构成比方面:与对照组相比,低剂量组中Ⅰ级精子比例升高,而在中、高剂量组下降;Ⅱ、Ⅲ级精子比例在低、高剂量组均降低;Ⅳ级精子比例在中、高剂量组上升;以上差异均具有统计学意义(P<0.05)。与对照组相比,各剂量组血清睾酮水平均升高(P<0.05),高剂量组的雌二醇水平下降(P<0.05)。相对于对照组,高剂量组的Hsd3β1Cyp19a1的mRNA表达下调(P<0.05),而StARCyp11a1的mRNA表达上调(P<0.05)。低、中、高剂量组的CYP11 A1蛋白表达量均高于对照组(P<0.05)。
    结论 妊娠期环境水平的砷暴露可诱导睾酮异常增高及降低子代雄鼠精子质量,是影响雄性后代生殖健康的潜在危险因素。

     

    Abstract:
    Background Arsenic is recognized as a kind of developmental toxicant, which can pass through the placenta barrier and induce health defects in offspring. However, the effects of environmental levels of arsenic exposure during gestation on the reproductive system of adult male offspring remain unclear.
    Objective To investigate the impact of environmental levels of arsenic exposure during gestation on testosterone synthesis and sperm quality in F1 adult male rats.
    Methods Forty sexually mature Wistar female rats were randomly divided into four groups according to body weight, namely control group, low-dose sodium arsenite (NaAsO2) group, middle-dose NaAsO2 group, and high-dose NaAsO2 group. They were mated with sexually mature Wistar male rats in a ratio of 2:1, and the day with presence of a vaginal plug or spermatozoa in the vaginal smear was designated as gestational day 0 (GD0). Pregnant rats were provided drinking water containing 0, 1, 5,, or 25 mg·L−1 NaAsO2 until delivery. At postnatal day 70, the F1 male rats were euthanized. Anogenital distance was measured, testis and epididymis were weighed, and associated organ coefficients were calculated. Epididymal sperm quality was evaluated. The histological changes of testis were observed. The levels of testosterone and estradiol in serum were determined with ELISA kit. The testicular mRNA relative expression levels of key steroidogenic enzymes were determined by quantitative real-time PCR. The protein relative expression levels of key steroidogenic enzymes were determined by Western blotting.
    Results Compared with the control group, the testicular coefficients and epididymis coefficients were increased in the low- and middle-dose groups (P<0.05), and the epididymis coefficient was also increased in the high-dose group (P<0.05). As for the percentage of sperm motility, compared to the control group, grade Ⅰ sperm cells were increased in the low-dose group, but significantly decreased in the middle- and high-dose groups; grade Ⅱ and Ⅲ sperm cells were decreased in the low- and high-dose groups; grade Ⅳ sperm cells were significantly increased in the middle- and high-dose groups; all the differences above were statistically significant (P<0.05). Compared with the control group, there was a significant increase in serum testosterone levels in all treated groups (P<0.05), and the serum estradiol levels were significantly decreased in the high-dose group (P<0.05). Meanwhile, compared with the control group, the relative mRNA expression levels of Hsd3β1 and Cyp19a1 were decreased (P<0.05), while those of StAR and Cyp11a1 were increased in the high-dose group (P<0.05). In addition, the relative protein expression levels of CYP11A1 were significantly increased in all treated groups compared with the control group (P<0.05).
    Conclusion Environmental levels of arsenic exposure during gestation can up-regulate testosterone level and reduce sperm quality, and is a potential risk for reproductive dysfunction in adult male offspring.

     

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