The effect of neonatal exposure to diethylstilbestrol (DES), a potent synthetic estrogen, was examined to evaluate whether the CD-1 (estrogen insensitive, outbred) and C57 (estrogen sensitive, inbred) mouse strains differ in their response to estrogen disruption of male ExG differentiation. CD-1 and C57BL/6 litters were injected with sesame oil or DES (200 ng/g/5 μl in sesame oil vehicle) every other day from birth to day 10. Animals were sacrificed at the following time points: birth, 5, 10 and 60 days postnatal. Neonatally DES-treated mice from both strains had many ExG abnormalities that included the following: (a) severe truncation of the prepuce and glans penis, (b) an abnormal urethral meatus, (c) ventral tethering of the penis, (d) reduced os penis length and glans width, (e) impaired differentiation of cartilage, (f) absence of urethral flaps, and (g) impaired differentiation of erectile bodies. Adverse effects of DES correlated with the expression of estrogen receptors within the affected tissues. While the effects of DES were similar in the more estrogen-sensitive C57BL/6 mice versus the less estrogen-sensitive CD-1 mice, the severity of DES effects was consistently greater in C57BL/6 mice. We suggest that many of the effects of DES, including the induction of hypospadias, are due to impaired growth and tissue fusion events during development.

Potential trans-generational influence of diethylstilbestrol (DES) exposure emerged with reports of effects in grandchildren of DES-treated pregnant women and of reproductive tract tumors in offspring of mice exposed in utero to DES. Accordingly, we examined the trans-generational influence of DES on development of external genitalia (ExG) and compared effects of in utero DES exposure in CD-1 and C57BL/6 mice injected with oil or DES every other day from gestational days 12 to 18. Mice were examined at birth, and on 5-120 days postnatal to evaluate ExG malformations. Of 23 adult (>60 days) prenatally DES-exposed males, features indicative of urethral meatal hypospadias (see text for definitions) ranged from 18% to 100% in prenatally DES-exposed CD-1 males and 31% to 100% in prenatally DES-exposed C57BL/6 males. Thus, the strains differed only slightly in the incidence of male urethral hypospadias. Ninety-one percent of DES-exposed CD-1 females and 100% of DES-exposed C57BL/6 females had urethral-vaginal fistula. All DES-exposed CD-1 and C57BL/6 females lacked an os clitoris. None of the prenatally oil-treated CD-1 and C57BL/6 male and female mice had ExG malformations. For the second-generation study, 10 adult CD-1 males and females, from oil- and DES-exposed groups, respectively, were paired with untreated CD-1 mice for 30 days, and their offspring evaluated for ExG malformations. None of the F1 DES-treated females were fertile. Nine of 10 prenatally DES-exposed CD-1 males sired offspring with untreated females, producing 55 male and 42 female pups. Of the F2 DES-lineage adult males, 20% had exposed urethral flaps, a criterion of urethral meatal hypospadias. Five of 42 (11.9%) F2 DES lineage females had urethral-vaginal fistula. In contrast, all F2 oil-lineage males and all oil-lineage females were normal. Thus, prenatal DES exposure induces malformations of ExG in both sexes and strains of mice, and certain malformations are transmitted to the second-generation.