No abstract

Background/Aims: Synthetic estrogens induce hypospadias, an anomaly of genital tubercle/urethral development. Activating transcription factor 3 (ATF3), which is estrogen-responsive in vitro, is upregulated in hypospadiac human tissue. We used a mouse model of steroid-dependent genital tubercle development to elucidate the ontogeny of ATF3 expression and the developmental response of ATF3 in vivo to estrogen exposure. Methods: We used quantitative RT-PCR to assess ontogenic expression of ATF3 and its response to estrogen treatment in utero. Immunohistochemistry was used to localize the protein. Results: Quantitative RT-PCR showed that ATF3 mRNA is upregulated in all estrogen-exposed fetal genital tubercles compared to controls (p = 0.024), including specifically in males exposed in utero (p = 0.049). Additionally, its expression increases significantly during the period of sexual differentiation in both sexes and significantly correlates with female development (p = 0.004), a phenomenon that appears to be attributable to higher levels at birth in females. The protein localizes in the nucleus, as expected. Conclusions: ATF3 is estrogen-responsive in vivo. The response of ATF3 to estrogenic stimulation in utero at an earlier stage may contribute to urethral abnormalities observed in estrogen-exposed male fetuses, although it is likely not the only gene involved, which supports the general understanding that hypospadias is subject to multifactorial influences. ATF3 may therefore be an appropriate gene for further investigations in an endocrine context.

In utero exposure to over-the-counter loratadine syrup can result in hypospadias in this model, and creates changes in the steroid receptor mRNA expression profile similar to those elicited by a synthetic estrogen.

We have used a mouse model to demonstrate that synthetic estrogens can induce hypospadias, an anomaly of genital tubercle/urethral development that may be attributable in part to the action of endocrine-disrupting compounds. In addition, we have found that activating transcription factor 3 (ATF3) is upregulated in hypospadiac human tissue. To further explore the potential involvement of ATF3 in hypospadias and its response to estrogen, we used a mouse model of steroid-dependent genital tubercle development to elucidate the ontogeny of ATF3 expression and to determine whether or not ATF3 responds developmentally in vivo to estrogen exposure.METHODS: 17-a-ethinyl estradiol (Sigma) at 50 f-Ig/kg was feed by oral gavage to timed pregnant mice on GDs 12-17. Controls received the oil vehicle only. Fetuses were removed n embryonic day 18 and the sex of each fetal mouse was determined by gonadal analysis. Genital tubercle tissues were fixed in formalin, paraffin embedded, and serially sectioned for histologic staining or were put into RNAiater (Qiagen) for purification total RNA. Immunohistochemical staining was used to check expression of ATF3 protein in genital tubercle tissue. Expression of ATF3 mRNA was studied by TaqMan realtime PCR (UCSF Cancer Center Comprehensive Genome Analysis Core). For statistical analysis, realtime PCR data were analyzed based on a median test for nonparametric data.RESULTS: Our results with quantitative RT-PCR show that ATF3 mRNA is upregulated in all estrogen-exposed fetal genital tubercles compared to controls (p=0.024), including specifically in males in response to in utero estrogen exposure (p=0.049). Additionally, its expression increases significantly during the period of sexual differentiation in both sexes and shows a significant correlation with female development (p=0.004), a phenomenon that appears to be attributable to higher levels at birth in females. Immunohistochemical results show that ATF3 protein localizes in the cell nucleus.CONCLUSIONS: We suggest that ATF3 may play a role in normal female sex differentiation and that its response to estrogenic stimulation in utero at an earlier stage may contribute to urethral abnormalities observed in estrogen-exposed male fetuses. ATF3 may therefore be an appropriate gene for further investigations in an endocrine-disruption context.