己烯雌酚致大鼠腹腔内隐睾及其对相关基因表达的影响

The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats

目的观察不同剂量己烯雌酚（DES）致大鼠腹腔内隐睾及其对睾丸胰岛素样因子3（INSL3）、引带同源盒基因-A10（HOXA10）表达的影响。方法将50只SD孕鼠按体重分层随机化的方法分为5组，每组10只，在孕期第13．5天（E13．5）分别皮下注射二甲基亚砜（A组）和DES2．5、5．0、10．0、20．0mg／kg（分别为B、C、D、E组），在第19．5天（E19．5）取出胚胎雄鼠，体视显微镜下观察后计算死胎率、睾丸下降程度；RT-PCR检测INSL3和HOXA10 mRNA水平，Western blotting检测INSL3蛋白水平。结果A、B、C、D和E组死胎率分别为3．57％、6．90％、12．00％、19．23％和36．36％，与DES呈量效关系（r=0．999，P〈0．01）；B、C、D和E组的睾丸下降程度分别为（23．7±1．7）U、（38．8±1．9）U、（49．3±1．8）U、（58．6±2．1）U，与A组[（8．5±1．3）U]相比，差异有统计学意义（q值分别为46．12、88．53、120．44、141．37，P值均〈0．01），且呈量效关系（r=0．911，P〈0．05）。B、C、D和E组的INSL3mRNA水平分别为0．9570±0．1490、0．6760±0．1380、0．0170±0．0040、0．0013±0．0003，蛋白表达水平分别为0．8360±0．1520、0．5310±0．1070、0．0140±0．0020、0．0011±0．0003，与A组（INSL3 mRNA和蛋白表达分别为1.8010±0．1260和1．6120±0．1340）相比，差异有统计学意义（qmRNA值分别为40．4840、52．4402、83．1585、82．0582，q蛋白值分别为38．6151、52．2747、77．2756、76．1983，P值均〈0．01）。A、B、C、D和E组的HOXA10 mRNA水平分别为0．945±0．125、0．940±0．119、0．656±0．115、0．544±0．118和0．463±0．114，A组与B组差异无统计学意义（q=0．2213，P〉0．05），A组与C～E组差异有统计学意义（q值分别为12．4304、17．2477、20．2789，P值均〈0．01）。结论DES致腹腔内睾丸下降受阻与其下调睾丸INSL3表达相关。低剂量DES未影响引带HOXA10表达，高剂量DES诱导的腹腔内隐睾与其下调HOXA10的表达相关。

Objective To study the effect of diaethylstilbestrol （DES） at different doses on transabdomial testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum. Method Fifty E13.5（ embryonic day 13.5） pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2. 5,5.0, 10. 0 and 20. 0 mg/kg DES （group A, B, C, D and E）, respectively. Male offspring were killed at E19. 5, and then fetal mortality, the degree of transabdominal testicular ascent （DTA） was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting. Results Male fetal mortality in group A, B, C, D, and E were 3.57% ,6. 90%, 12.00%, 19. 23% and 36. 36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality （ r = 0. 999, P 〈 0. 01 ）. DTA in group B, C, D and E were （23.7 ±1.7） U, （38. 8± 1.9） U, （49.3 ± 1.8） U and （58.6±2. 1 ） U that were significantly larger than that in group A [ （ 8.5 ± 1.3 ） U ] （ q = 46. 12,88.53,120.44 and 141.37, respectively, P 〈 0. 01）.There was also a dose-effect relationship between DES and DTA. In group B, C, D, and E, the expression of INSL3 mRNA were 0. 9570 ± 0. 1490,0. 6760 ± 0. 1380,0. 0170 ± 0. 0040 and 0. 0013± 0. 0003 ,respectively;the expressions of INSL3 protein were 0. 8360 ± 0. 1520,0. 5310 ± 0. 1070,0. 0140 ± 0. 0020 and 0. 0011±0. 0003, respectively, which were significantly larger than the expression of INSL3 mRNA （1. 801 ± 0. 126） and INSL3 protein （1. 612 ± 0. 134 ） in group A （qmRNA = 40. 4840,52. 4402, 83. 1585 and 82. 0582 ,respectively ,and qprotein = 38. 6151 ,52. 2747,77. 2756 and 76. 1983 ,respeetively,P 〈 0. 01）. The expression of HOXA10 mRNA in group A,B,C,D,and E were 0. 945±0. 125,0. 940 ±0. 119, 0. 656 ± 0. 115,0. 544 ±0. 118 and 0. 463±0. 114, respectively. Compared with the expression of HOXA10 mRNA in group A ,the expression of group B was not significantly different （q = 0. 2213 ,P 〉 0. 05 ） ,those in other groups were down-regulated significantly （ q = 12. 4304, 17.2477 and 20. 2789, respectively, P 〈 0. 01 ）. Conclusion DES inhibited transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal eryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.