Crucial Roles of the Mesenchymal Androgen Receptor in Wolffian Duct Development

Abstract

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.

Keywords: Wolffian duct; androgen receptor; androgens; epididymis; mesenchyme.

Figure 1.

Mesenchyme-specific ablation of Ar leads to defective Wolffian duct morphogenesis. A, Immunofluorescent staining of DsRed for tdTomato expression in Osr2^Cre+; Rosa^tdTomato+ male embryos at 3 time points (E11.5, E12.5, and E14.5). Dashed white lines demarcate Wolffian duct epithelium, which is negative for tdTomato expression. Scale bar: 25 μm. B, Immunohistochemical staining of androgen receptor (AR) at E14.5. Blue arrows: Wolffian duct epithelium. Scale bar: 50 μm. C, Brightfield image of PND0 control and AR^cKO mice. Scale bar: 4.0 mm.

Figure 2.

Visualization of Wolffian duct development over time in control and AR^cKO groups. Ex vivo organ culture of paired male reproductive tracts with testes attached in control and AR^cKO groups starting at E14.5 (day 0) for 4 days. Blue and purple arrows indicate cranial and caudal Wolffian duct, respectively. Scale bar: 2.0 mm.

Figure 3.

Attenuated ductal growth and cellular proliferation in AR^cKO Wolffian ducts. A and B, Quantification of the Wolffian duct circumference (μm) A, and the average number of epithelial cells per section, B, in the control and AR^cKO groups. C, Immunofluorescent staining of cell proliferation marker Ki67 and cell apoptotic marker cleaved-PARP1 in the control group and AR^cKO groups at E14.5. Scale bar: 25 μm. Dashed blue lines outline Wolffian duct epithelial layers. D and E, Quantifications of the percentage of proliferating and apoptotic cells in Wolffian duct epithelium and mesenchyme in control AR^cKO groups. Results were shown as mean ± SD and analyzed by 2-tailed t test. NS, no significance. P less than .05; *P less than .05 compared to the control group. N = 5 for each group.

Figure 4.

Identification of differentially expressed genes and pathways by RNA sequencing in AR^cKO mesonephroi. A, Volcano plot displaying gene expression at E14.5. Differentially expressed genes (DEGs; adjusted P values <.1) in the AR^cKO group are highlighted in blue (downregulated genes) and red (upregulated genes), respectively. B, Gene ontology (GO) analysis showing enriched cellular components and biological processes associated with DEGs. C, Dot plots illustrating the expression levels of 4 selected genes (Ar, Top2a, Wnt9b, and Lama2). All 4 genes were significantly downregulated in the AR^cKO group. TMP, transcripts per million.

Figure 5.

Expression of Wnt9b and laminin in control and AR^cKO mesonephroi. Wnt9b and Laminin expression were localized by RNAscope and immunofluorescent staining, respectively. Blue arrows indicate Wolffian ducts. Scale bar: 50 μm.

Figure 6.

Reduced smooth muscle differentiation in Wolffian ducts of AR^cKO male embryos. A, Immunofluorescent staining of the smooth muscle marker alpha-smooth muscle actin (αSMA) in the control group and AR^cKO group at E16.5. Blue dashed lines outline Wolffian duct epithelium. Scale bar: 50 μm. B, Quantifications of the average area (μm²) of αSMA+ smooth muscle cells in the control and AR^cKO groups. Results were shown as mean ± SD. and analyzed by 2-tailed unpaired t test. *P less than .05 compared to the control group. N = 5 for each group.