[Hypospadias induced by maternal exposure to di-n-butyl phthalate and its mechanisms in male rat offspring , , , , ,]

To induce hypospadias in male rat offspring by maternal exposure to di-n-butyl phthalate (DBP) during late pregnancy and further investigate its mechanisms.We randomly divided 20 pregnant rats into a DBP exposure and a control group, the former treated intragastrically with DBP while the latter with soybean oil at 750 mg per kilogram of the body weight per day from gestation days (GD) 14 to 18. On postnatal day (PND) 1, we recorded the incidence rate of hypospadias and observed the histopathological changes in the genital tubercle of the hypospadiac rats. We also measured the level of serum testosterone (T) by radioimmunoassay and determined the mRNA and protein expressions of the androgen receptor (AR), sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4) and fibroblast growth factor 8 (Fgf8) in the genital tubercle by real-time PCR and Western blot.No hypospadiac male rats were found in the control group. The incidence rate of hypospadias in male offspring was 43.6% in the DBP-treatment group. Histological analysis confirmed hypospadiac malformation. The serum testosterone concentration was decreased in the hypospadiac male rats as compared with the controls (［0.49 ± 0.05］ vs ［1.12 ± 0.05］ ng/ml, P0.05). The mRNA expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle were significantly lower in the hypospadiac male rats than in the controls (AR: 0.50 ± 0.05 vs 1.00 ± 0.12, P0.05; Shh: 0.65 ± 0.07 vs 1.00 ± 0.15, P0.05; Bmp4: 0.42 ± 0.05 vs 1.00 ± 0.13, P0.05; Fgf8: 0.46 ± 0.04 vs 1.00 ± 0.12, P0.05), and so were their protein expressions (AR: 0.34 ± 0.05 vs 1.00 ± 0.09, P0.05; Shh: 0.51 ± 0.07 vs 1.00 ± 0.12, P0.05; Bmp4: 0.43 ± 0.05 vs 1.00 ± 0.11, P0.05; Fgf8: 0.57 ± 0.04 vs 1.00 ± 0.13, P0.05).Maternal exposure to DBP during late pregnancy can induce hypospadias in the male rat offspring. DBP affects the development of the genital tubercle by reducing the serum T concentration and expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle, which might underlie the mechanism of DBP inducing hypospadias.目的： 邻苯二甲酸二丁酯（DBP）孕晚期染毒诱导雄性SD大鼠子鼠发生尿道下裂畸形，并进一步探讨DBP致雄性子鼠发生尿道下裂的分子作用机制。方法： 孕鼠20只，怀孕14~18 d，随机分为对照组和DBP染毒组，每天分别予大豆油及DBP 750 mg/kg灌胃。出生后（PND）1 d，统计雄性子鼠中尿道下裂的发生率，观察尿道下裂雄性子鼠生殖结节的病理学改变。放射免疫法检测血清雄激素水平，实时荧光定量PCR和Western印迹检测生殖结节中雄激素受体（AR）、音猬因子（Shh）、骨形态发生蛋白4（Bmp4）和成纤维细胞成长因子8（Fgf8）的mRNA和蛋白表达水平。结果: 对照组中无尿道下裂发生，DBP染毒组中雄性子鼠尿道下裂发生率为43.6%,病理学检查发现生殖结节典型的尿道下裂畸形。尿道下裂雄性子鼠的血清雄激素水平［（0.49±0.05) ng/ml］较对照组［（1.12±0.05) ng/ml］明显降低（P0.05），生殖结节中AR、Shh、Bmp4和Fgf8基因的mRNA水平（0.50±0.05、0.65±0.07、0.42±0.05、0.46±0.04）较对照组（1.00±0.12、1.00±0.15、1.00±0.13、1.00±0.12）明显降低（P0.05），蛋白水平（0.34±0.05、0.51±0.07、0.43±0.05、0.57±0.04）较对照组（1.00±0.09、1.00±0.12、1.00±0.11、1.00±0.13）明显降低（P0.05）。结论： 孕晚期DBP染毒可以导致雄性子鼠发生尿道下裂畸形。DBP通过降低雄性子鼠血清雄激素水平和生殖结节中AR、Shh、Bmp4、Fgf8基因的表达，影响生殖结节的正常发育，是DBP导致尿道下裂畸形发生的可能机制。.