Androgen-regulated gene expression of developing external genitalia in mice

Androgen signaling during a critical embryonic window is essential for male embryonic external genitalia (eExG) development, including urethral tubularization, and defects in the androgen signaling pathway result in common congenital anomalies, such as hypospadias. To identify key regulators for eExG differentiation, we manipulated the hormonal environment of developing mouse embryos by administering testosterone propionate (TP) to pregnant dams to induce masculinization in female embryos or the androgen receptor (AR) antagonist flutamide (FLU) to induce feminization in male embryos. We performed a transcriptome analysis (RNA-seq) on eExG of E16.5 male and female embryos derived from the control and hormonally treated dams. Principal component analysis of the gene expression profiles revealed sexually intermediate gene expression patterns in TP-treated females and FLU-treated males. Based on differential gene expression analysis between control males versus females, control males versus FLU-treated males, and TP-treated females versus control females, we identified 58 male-associated and 78 female-associated genes as candidate key regulators for eExG sexual differentiation. Gene ontology analysis of these 136 candidates highlighted enrichment for mesenchymal development and organization. However, only a few of these genes contained canonical androgen response elements, suggesting frequent indirect AR regulation. By analyzing gene expression under manipulated hormonal conditions, we successfully identified candidate genes strongly associated with eExG masculinization and feminization. Our findings highlight the critical role of mesenchymal development and organization, and provide a valuable gene set for future studies on eExG development and disorders of sex development.