Visser J A, McLuskey A, Verhoef-Post M, Kramer P, Grootegoed J A, Themmen A P
Department of Endocrinology and Reproduction, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, The Netherlands.
Endocrinology. 1998 Oct;139(10):4244-51. doi: 10.1210/endo.139.10.6215.
The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daughters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Müllerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Müllerian ducts is under testicular control through anti-Müllerian hormone (AMH). In male mice, treated in utero with DES, the Müllerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Müllerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Müllerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied. It was observed that Müllerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during pregnancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Müllerian duct formation by approximately 2 days; in untreated fetuses, Müllerian duct formation was complete by E13, whereas fully formed Müllerian ducts were not observed in DES-treated male fetuses until E15. Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E 13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal development. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Müllerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Müllerian duct development, which may result in an asynchrony in the timing of Müllerian duct formation, with respect to the critical period of Müllerian duct regression, leading to persistence of Müllerian duct remnants in male mice.
孕妇使用己烯雌酚（DES）导致这些孕期所生女儿的生殖道癌发病率增加。此外，在所谓的DES儿子中也发现了异常，主要是存在苗勒管残余物，这表明胎儿接触DES可能对男性性别分化有影响。苗勒管的胎儿期退化受睾丸通过抗苗勒管激素（AMH）的控制。在子宫内用DES处理的雄性小鼠中，尽管接触DES的睾丸确实产生AMH，但苗勒管并未完全退化。我们假设，接触DES的雄性小鼠中苗勒管退化不完全是由于苗勒管对AMH的敏感性降低所致。因此，研究了DES对雄性小鼠胎儿苗勒管退化的时间方面以及AMH II型受体（AMHRII）信使核糖核酸（mRNA）表达的影响。观察到，在孕期从E9至E16给予DES后，E19（交配后19天）时苗勒管退化不完全。此外，对胎儿发育早期时间点的分析表明，DES处理明显使苗勒管形成的起始延迟了约2天；在未处理的胎儿中，E13时苗勒管形成完成，而在DES处理的雄性胎儿中，直到E15才观察到完全形成的苗勒管。使用原位杂交，在接触DES的雄性胎儿中未观察到AMH和AMHRII mRNA表达定位的变化。使用核糖核酸酶保护测定法定量mRNA表达，结果显示接触DES的雄性胎儿在E13时AMH和AMHRII mRNA的表达水平增加。此外，测定了Hoxa 11和类固醇生成因子-1（SF-1）的mRNA表达水平作为胎儿发育的标志物。产前接触DES对Hoxa 11 mRNA表达没有影响，表明DES对胎儿发育速度没有整体影响。在接触DES的雄性胎儿中，SF-1的mRNA表达增加情况与AMH相似，这与以下观察结果一致，即AMH基因启动子需要完整的SF-1 DNA结合位点才能进行时间和细胞特异性表达，尽管不能排除DES对肾上腺和垂体等其他组织中SF-1表达的影响。然而，AMH和AMHRII mRNA表达水平的增加并不能直接解释苗勒管对AMH敏感性降低的原因。因此，得出结论，雄性小鼠产前接触DES会延迟苗勒管发育的起始，这可能导致苗勒管形成时间与苗勒管退化关键期不同步，从而导致雄性小鼠中苗勒管残余物的持续存在。
