EFFECT OF PRENATAL EXPOSURE TO DIETHYLSTILBESTROL ON MULLERIAN DUCT DEVELOPMENT IN FETAL MALE-MICE

The clinical use of diethylstilbestrol (DES) by pregnant women has res ulted in an increased incidence of genital carcinoma in the daughters born from these pregnancies. Also, in the so-called DES-sons abnormali ties were found, mainly, the presence of Mullerian duct remnants, whic h indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Mullerian ducts is under test icular control through anti-Mullerian hormone (AMH). In male mice, tre ated in utero with DES, the Mullerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that inco mplete regression in DES-exposed males is caused by a diminished sensi tivity of the Mullerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Mullerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was st udied. It was observed that Mullerian duct regression was incomplete a t E19 (19 days post coitum), upon DES administration during pregnancy from E9 through E16. Furthermore, analysis of earlier time points of f etal development revealed that the DES treatment had clearly delayed t he onset of Mullerian duct formation by approximately 2 days; in untre ated fetuses, Mullerian duct formation was complete by E13, whereas fu lly formed Mullerian ducts were not observed in DES-treated male fetus es until E15. Using in, situ hybridization, no change in the localizat ion of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protec tion assay, showing an increased expression level of AMH and AMHRII mR NAs at E13 in DES-exposed male fetuses. Furthermore, the mRNA expressi on levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effec t on Hoxa 11 mRNA expression, indicating that DES did not exert an ove rall effect on the rate of fetal development. In DES-exposed male fetu ses, SF-l showed a similar increase in mRNA expression as AMH, in agre ement with the observations that the AMH gene promoter requires an int act SF-1 DNA binding site for time- and cell-specific expression, alth ough an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increas ed expression levels of AMH and AMHRII mRNAs do not directly explain t he decreased sensitivity of the Mullerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Mullerian duct development, which may result in an asynchrony in th e timing of Mullerian duct formation, with respect to the critical per iod of Mullerian duct regression, leading to persistence of Mullerian duct remnants in male mice.