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 to 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 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.

Dgkk appears to be a marker or mediator of squamous cell differentiation during development of mouse external genitalia. However, no association exists between Dgkk expression and formation of preputial cleft in the genital tubercle of diethylsilbestrol treated mice, suggesting that these 2 events may follow independent pathways in mice. Further studies are needed to elucidate the role of DGKK in hypospadias.

During muscle development, the laminin-specific ␣7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the ␣7X1 or the ␣7X2 variant. The relative level of ␣7X1 and ␣7X2 is developmentally regulated. Similarly, the partner ␤1 integrin cytoplasmic domain is converted from the ␤1A to the ␤1D splice variant. To determine whether ␤1D modulates the activity of the ␣7 receptor, cells were transfected with ␣7X1 and ␤1D cDNA. ␣7X1 coupled with ␤1A failed to adhere to laminin-1, whereas cotransfectants expressing ␣7X1 and ␤1D showed strong adhesion. Interestingly, ␣7X1 complexed with ␤1A and ␤1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that ␣7 function is regulated not only by X1/X2 in its extracellular domain but also by ␤1 cytoplasmic splice variants. It is likely that expression of ␤1D alters ␣7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of ␣7␤1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.