邻苯二甲酸二丁酯影响类固醇激素合成急性调节蛋白致雄性胎鼠尿道下裂的机制研究

Differential expression and activity of steroidogenic acute regulatory protein in testes of hypospadiac male rats with in utero exposure to di-n-butyl phthalate

目的 研究类固醇激素合成急性调节蛋白（StAR）的表达和活性及睾酮在尿道下裂胎鼠体内发生的改变，探讨邻苯二甲酸二丁酯（DBP）诱导雄性胎鼠发生尿道下裂的机制。方法 将40只孕SD大鼠，用随机数表法分为DBP组和对照组（各20只）。于孕13～19 d上午9点给药，DBP组按照800 mg/kg体重DBP混合玉米油（DBP和玉米油总量为3 ml/kg体重）灌胃，对照组按3 ml/kg体重玉米油灌胃。第19天中午12时麻醉孕鼠后取出胎鼠，观察内外生殖器鉴别出DBP组发生尿道下裂的雄性胎鼠及对照组中的雄性胎鼠。从DBP组中发生尿道下裂的雄性胎鼠中选取40只做为尿道下裂组，再从对照组的雄性胎鼠中选取40只做为正常对照组，拍摄尿道下裂图片，测量肛门生殖结节距离，统计尿道下裂发生率，取各组胎鼠生殖结节进行HE染色。各组胎鼠断头取血用酶联免疫吸附测定法（ELISA）检测睾酮水平并取出各组睾丸采用定量逆转录聚合酶链式反应（RT-PCR）、Western blot、免疫荧光、ELISA检测StAR mRNA及StAR的表达，对StAR相对表达量和睾酮含量进行Pearson相关性分析。Western blot检测磷酸化ERK1/2蛋白（P-ERK1/2），总ERK1/2蛋白（T-ERK1/2）的表达，并通过两者间的比值分析睾丸中ERK1/2蛋白的磷酸化水平以明确StAR蛋白的活性高低。结果 尿道下裂组胎鼠的尿道开口于生殖结节腹侧，对照组的开口于生殖结节顶端。尿道下裂组肛门生殖结节距离（1.77±0.12） mm明显小于对照组（2.25±0.15） mm，组间比较，差异有统计学意义（P〈0.05）。DBP组胎鼠尿道下裂发生率为43.3%（46/106）。尿道下裂组睾酮含量（1.45±0.62） ng/ml低于对照组（4.48±0.93） ng/ml，组间比较，差异有统计学意义（P〈0.05）。尿道下裂组的StAR mRNA、StAR相对表达量及P-ERK1/T-ERK1、P-ERK2/T-ERK2分别为0.23±0.08、0.33±0.07、0.17±0.03和0.19±0.07，对照组的分别为1.00±0.00、1.44±0.19、0.31±0.08和0.43±0.14，组间差异有统计学意义（P〈0.05）。免疫荧光显示睾丸间质细胞中尿道下裂组StAR蛋白的表达少于对照组。尿道下裂组和对照组的Pearson相关系数分别为0.642和0.851（P均〈0.05）。结论 DBP不仅在转录和翻译层面降低StAR的表达，可能还通过降低ERK1/2蛋白的磷酸化水平减弱StAR蛋白的活性使睾丸中睾酮合成障碍，进而影响了雄性胎鼠生殖器的发育。

Objective To explore the pathogenesis of di-n-butyl phthalate （DBP）-induced hypospadias, detect the differential expression and activity of steroidogenic acute regulatory （StAR） protein in fetal testes and measure the testosterone concentration in sera.Methods Forty pregnant Sprague Dawley rats were randomly divided into two groups （n=20 each）. Dams were treated by gavage at 9 a. m daily from gestation day （GD） 13 to GD19 with corn oil vehicle （3 ml/kg） or DBP in corn oil at 800 mg/kg （total DBP ＆ corn oil at 3 ml/kg）. On GD19 at 12 a. m, fetuses were immediately removed from uterus and sexed by internal examination of reproductive organs. Hypospadiac and normal rats were randomly divided into hypospadiac and control groups. Outer appearance of genital tubercles（GT） was examined. And anogenital distance （AGD） was measured. The incidence of hypospadias was calculated. And GT was stained with hematoxylin ＆ eosin （H＆E）. The concentration of testosterone was measured with enzyme-linked immunosorbent assay （ELISA） kits. Testes were removed from male fetuses. Real-time quantitative polymerase chain reaction （PCR）, Western blot, ELISA and immunofluorescence were used for measuring the expression of StAR mRNA or StAR protein in testes. Pearson＇s correlation was used for analyzing the relationship between relative expression of StAR protein and testosterone concentration. Western blot was utilized for quantifying the expression of T-ERK1/2 and P-ERK1/2 in testes. To determine the activity of StAR protein, ERK1/2 phosphorylation was analyzed by the ratio of P-ERK1/2/T-ERK1/2.Results H＆E stain showed that urethral orifice of hypospadias was located on ventral surface of GT and urethral orifice of normal rats appeared adjacent to the tip of GT. AGD was significantly lower in hypospadiac group than that in control group [（2.25±0.15） vs. （1.77±0.12）, P〈0.05]; incidence of hypospadias at 43.3%（46/106）. The testosterone concentration was also lower in hypospadiac group than that in control group [（1.45±0.62） ng/ml vs. （4.48±0.93） ng/ml, P〈0.05]. Significant down-regulation of StAR was also found in both mRNA and protein in hypospadiac group （StAR mRNA： 0.23±0.08; StAR： 0.33±0.07） when compared with control group （StAR mRNA： 1.00±0.00; StAR： 1.44±0.19, P〈0.05）. StAR protein was located predominantly in interstitial cell of tests and staining intensity was obviously weaker in hypospadiac group than that in control group. Pearson’s correlation coefficient was also lower in hypospadiac group （r=0.642, P〈0.05） than that in control group （r=0.851, P〈0.05）. The ratio of P-ERK1/2/ERK1/2 （P-ERK1/ERK1： 0.17±0.03; P-ERK2/ERK2： 0.19±0.07） significantly decreased in hypospadiac group as compared with control group （P-ERK1/ERK1： 0.31±0.08; P-ERK2/ERK2： 0.43±0.14, P〈0.05）.Conclusions DBP not only decreases the expression of StAR at the levels of gene and protein translation, but also inhibits the activity of StAR protein by decreasing the level of ERK1/2 phosphorylation to affect the synthesis of testosterone, thereby inducing hypospadias.