Methods: Pregnant Sprague-Dawley rats were given corn oil or corn oil plus 17-α-ethinyl-estrogen at embryonic days (ED) 12-17. At ED 18, embryos from treated or untreated dams were examined for lack of sulcus closure in the genital tubercle and cultured in DMEM, 3 days. Human foreskin fibroblasts (BJ cells) were treated every 24 hours with 100 nM of Diethylstibestrol (DES) for 6, 48 and 120 hours +/- 2-dexoxy-5-azacytidine (aza) or shRNA against DNMTs, to inhibit DNA methylation. RNA was extracted from cells and tissues for real-time PCR to query expression of candidate genes (WNT5A, HoxA13, DNA methyltransferases (DNMT-1, -3A and -3B, and others) vs housekeeping genes (rpl19, gapdh) using the deltadeltac(t) method. Protein was examined by western blotting. Live cell microscopy of development of the GT with and without estrogen exposure was also performed in myristolyated Venus CD-1 embryos
Results: In foreskin fibroblasts, expression of Wnt5A at 48 hours, and HoxA13 at 120 hours, in cells was downregulated by DES and recovered by azacytidine treatment, p<0.05. Wnt5A expression showed a trend in downregulation in cultured GTs from dams treated with estrogen, p=0.2. β-catenin pathway genes were also downregulated by estrogen treatment, including FN1 p<0.02. Intercalation and migration of cells in subepithelial layers of developing GTs were visualized in embryos at E12.5 to E17.5. Furthermore, in utero estrogen treatment appeared to inhibit migration patterns of cells in estrogen-treated embryos.
Conclusion: Estrogen may regulate methylation-dependent candidate gene expression and crucial cell biological traits in the developing genital tubercle.