Expression Analysis of DGKK during External Genitalia Formation.

PURPOSE: Genetic variants in diacylglycerol kinase κ (DGKK) have been strongly associated with risk of hypospadias. We investigated the expression pattern of Dgkk during development of mouse external genitalia to better understand its function and mechanism in the etiology of hypospadias. MATERIALS AND METHODS: We performed Dgkk expression analysis via indirect immunofluorescence in histological sections of CD-1 mouse embryonic and postnatal male, female and diethylsilbestrol treated external genitalia. Histological findings were supplemented with DGKK expression analysis using quantitative real-time polymerase chain reaction assays. RESULTS: In mouse external genitalia Dgkk was expressed in the membrane and cytoplasm of differentiating squamous epithelial cells of urethral plate/groove and epidermis but not in the undifferentiated epithelial cells of preputial lamina or basal layer of urethral groove epithelium. CD-1 gestation day 18 male mouse genital tubercle treated with oil or diethylstilbestrol showed similar patterns of Dgkk expression despite many morphological differences, including formation of preputial cleft observed in diethylsilbestrol treated mice. CONCLUSIONS: 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.

Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains.

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.

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin.

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.