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Epigenetic Mechanisms Direct Estrogen-Induced Downregulation of Gene Expression and Cell Biological Processes Critical for Genital Tubercle Formation

Saturday, October 20, 2012: 10:21 AM
Grand Ballroom A/B (Hilton Riverside)
Karen J. Aitken, B.Sc., Ph.D1, Jiaxin Jiang1, Matthew Bechbache, BSc, (pending)1, Tyler Kirwan1, Sevan Hopyan, MD, PhD, FRCSC2 and Darius Bagli3, (1)Developmental and Stem Cell Biology, Urology, Hospital for Sick Children, Toronto, ON, Canada, (2)Developmental and Stem Cell Biology, Research Institute; Orthopaedic Surgery, Department of Surgery, Hospital for Sick Children, Toronto, ON, Canada, (3)Division of Urology, Surgery; Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON, Canada

Purpose: Hypospadias is associated with in utero Xenoestrogen (XE) exposure (via diethylstilbesterol, vinclozidin, genistein and others), both clinically and experimentally. Conversely, many genes (SHH, WNT5A, β-catenin, HOXA13) are critical for genital tubercle (GT) and urethral development in mouse genetic models. The role of such genes in the response to XE exposure in GTs has been less studied, although XE exert known epigenetic effects. We hypothesized that estrogen exposure might result in altered expression of the aforementioned genes/pathways due to epigenetic regulation resulting in deficits in GT cell biology.

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.