Characterization of diethylstilbestrol-induced hypospadias in female mice

The urethral duct and vagina are formed from the urogenital sinus (UGS) during the early neonatal period in mice. Neonatal estrogen exposure results in hypospadias, or the malpositioning of vaginal and urethral openings, with wide cleft clitoris. We sought to characterize diethylstilbestrol (DES) influence on UGS morphogenesis and hypospadias formation. Newborn (day 0) and 1-4-day-old female mice (ICR/Jcl) were given (s.c.) oil or 3.0 microg DES. Animals were killed 24 hr later; then hypospadias formation and epithelial apoptosis and proliferation within the developing UGS were assessed. DES did not alter normal UGS morphogenesis by day 1, in comparison with controls. However, hypospadias formation was observed in DES-treated mice by day 3. In these mice, the distal dorsal urethral duct appeared to fuse with and open into the lower vaginal solid cord region. Further, DES treatment produced a gradual significant increase in dorsal urethral epithelial apoptosis (P < 0.05) just prior to and during fusion and hypospadias formation. DES-induced urethral epithelial and sinus cord proliferation appeared significantly increased (P < 0.05) and unchanged, respectively, just prior to fusion. By day 5, DES-treated mice exhibited wide cleft clitoris. In addition, if DES was given on day 3 or 5, a gradual, distinct caudal shift in the vaginal-urethral junction was observed compared to mice treated on days 0-2. Although hypospadias was not induced when neonates were given DES on day 7, these mice continued to display early vaginal opening. Dose-response analysis indicated that 0.03 microg DES for 5 days is the lowest known critical dose for hypospadias induction. We have shown for the first time that DES-induced hypospadias onset may primarily be the result of changes in developing dorsal urethral epithelial cell apoptotic and proliferative activity, and that the location of DES-induced hypospadias formation is dependent on age at time of exposure.