砷对大鼠附睾转化生长因子β1表达及精子顶体蛋白酶的影响

Effects of chronic exposure to sodium arsenic on expressions of TGF-β1 and sperm acrosin enzyme motility in epididymis of rats

目的研究慢性砷中毒对大鼠附睾转化生长因子β1(TGF-β1)及精子顶体蛋白酶的影响,为砷中毒导致的男性不育提供理论依据。方法健康清洁级雄性SD大鼠40只,体重160~200 g,随机分为高(60.0 mg/L)、中(12.0 mg/L)、低(2.4 mg/L)剂量砷染毒组及对照组(蒸馏水),采用经口自由饮用方式进行染毒,连续6个月。染毒结束后,采用免疫组织化学法、Western blotting及实时荧光定量聚合酶链反应(qRT-PCR)检测TGF-β1的表达变化,改良底物膜法观察精子顶体蛋白酶活性(阳性率和活性强度)。结果各组TGF-β1表达水平比较,差异有统计学意义(P<0.05),不同剂量砷染毒组与对照组两两比较,差异有统计学意义(P<0.05)。与对照组比较,不同剂量砷染毒组TGF-β1的蛋白及mRNA表达水平下调(P<0.05)。与对照组比较,低剂量组精子顶体蛋白酶活性差异无统计学意义(P>0.05);中、高剂量组精子顶体蛋白酶活性均下调(P<0.05)。结论慢性砷中毒对大鼠附睾TGF-β1的表达有影响,引起精子顶体酶活性下降,可能与男性不育有关。TGF-β1有望成为治疗慢性砷中毒引起男性不育的新靶点。

Objective To study the effects of chronic arsenic poisoning on the TGF-β1 and sperm acrosome enzyme motility in rats’ epididymis, and to provide theoretical basis for male infertility caused by arsenic poisoning. Methods Forty healthy and clean male SD rats, weighing about 160 to 200 g, were randomly divided into high(60.0 mg/L), medium(12.0 mg/L), low(2.4 mg/L) and control(distilled water) groups. After the inoculation, the expression of TGF-β1 was determined by immunohistochemistry, western blot and quantitative real-time PCR(qRTPCR), and the activities of sperm acrosomal enzyme(positive reaction rate of acrosomal enzyme;activity intensity of acrosomal enzymes) were observed by improved substrate membrane method. Results Immunohistochemical results showed that, compared with control group, the levels of expressions of TGF-β1 in infected group were lower, and the differences had statistical significance(P < 0.05). Compared between each two groups among them, the differences were statistically significant(P < 0.05). Western blotting and RT-PCR results showed that the protein and mRNA expression levels of TGF-β1 were significantly lower in each poisoned group(P < 0.05). Compared with the control group(positive rate of acrosin: 92.5 ± 11.5;activity intensity of acrosin: 50.3 ± 23.1), there was no significant difference in sperm acrosomal enzyme activity of the low dose group(positive rate of acrosin: 83.3 ± 10.6;activity intensity of acrosin: 37.4 ± 19.3)(P > 0.05);the sperm acrosomal enzyme activity in middle and high dose group(positive response rate of acrosomal enzyme: 40.0 ± 9.2;20.4 ± 6.4;activity intensity of acrosin: 16.8 ± 13.7, 8.9 ± 2.5) was significantly lower(positive rate: F = 760.000, P = 0.001;activity intensity: F = 27.760, P = 0.002). Conclusions Chronic arsenic poisoning has an effect on the expression of TGF-β1 in the epididymis of rats, and causes sperm acrosomal enzyme activity decreasing, which may be related to male infertility. TGF-β1 is expected to become a new target for the treatment of male infertility caused by chronic arsenic poisoning.