Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice

Abstract

The sexual differentiation paradigm contends that the female pattern of the reproductive system is established by default because the male reproductive tracts (Wolffian ducts) in the female degenerate owing to a lack of androgen. Here, we discovered that female mouse embryos lacking Coup-tfII (chicken ovalbumin upstream promoter transcription factor II) in the Wolffian duct mesenchyme became intersex-possessing both female and male reproductive tracts. Retention of Wolffian ducts was not caused by ectopic androgen production or action. Instead, enhanced phosphorylated extracellular signal-regulated kinase signaling in Wolffian duct epithelium was responsible for the retention of male structures in an androgen-independent manner. We thus suggest that elimination of Wolffian ducts in female embryos is actively promoted by COUP-TFII, which suppresses a mesenchyme-epithelium cross-talk responsible for Wolffian duct maintenance.

Fig. 1. Coup-tfII ablation leads to Wolffian duct retention in the XX embryo

Control and Coup-tfII knockout reproductive systems from E18.5 XX embryos were analyzed by whole mount immunofluorescence for the epithelial marker PAX2 (A & D) or AP-2a on frozen sections (B & E). Control and knockout mesonephroi from E18.5 XX embryos were cultured for 7 days and analyzed by bright field microscopy (C & F) or AP-2a immunofluorescence on frozen sections (insets in C & F). Blue arrows: Wolffian ducts; Magenta arrows: Müllerian ducts; White asterisk: ovary; ov= ovary; ut= uterus; va= vagina. Scales bars: 0.5 mm (A & D, C & F) and 50 µm (B, E and insets in C & F). N=14 in A; N=3 per each genotype in B, C, E, F, and N=23 in D).

Fig. 2. Wolffian duct retention in Coup-tfII knockout XX embryo is independent of androgen production or action

(A & B) mRNA expression of two rate-limiting steroidogenic enzymes, Hsd3b1 and Cyp17a1, in E14.5 control testis (light blue), control ovary (light pink), and knockout ovary (dark pink). (C) Anogenital distance (AGD) of the control and knockout embryos at E18.5. (D) PAX2 whole mount immunofluorescence of ovaries and mesonephroi after 4-day culture. N=7 per genotype. (E–G) mRNA expression of androgen receptor Ar and androgen-responsive genes (Slc26a3 and Folh1) in control XY, control XX, and knockout XX mesonephroi. Results are shown as mean ± SEM. Asterisks in A–C, F and G represent statistical significance of p<0.05 compared to either control or knockout XX samples by one-way ANOVA followed by Tukey’s test (N=8 per each group in A–B, N=8–11 in C; and N=8 in E–G). (H) Knockout XX embryos were exposed to either vehicle (oil) or androgen receptor antagonist flutamide in utero once daily from E12.5 to 17.5. Samples were collected at E18.5 and analyzed by PAX2 whole mount immunofluorescence. N=3 pe r genotype. Blue arrows: Wolffian ducts; Magenta arrows: Müllerian ducts. White asterisk: ovary. Scale bar in D & H: 0.5 mm.

Fig. 3. Enhanced FGF signaling is involved in Wolffian duct retention in the absence of Coup-tfII

(A) mRNA expression of FGF receptors and ligands in the mesonephroi at E14.5. UD: undetected. Results are shown as mean ± SEM. Asterisks represent statistical significance of p<0.05 compared to control females using Student’s t test (N=8 for each genotype). (B) Whole mount immunofluorescence of PAX2 of 2-day cultured E14.5 wild type XX mesonephroi in the presence of vehicle, or FGF7, or FGF10 or FGF7+FGF10. (C) p-ERK immunohistochemistry of control and knockout XX mesonephroi at E14.5. (D) PAX2 whole mount immunofluorescence in vehicle- or p-ERK inhibitor-treated knockout XX genital ridges. N=3 per group in B–D. Scale bars: 0.5 mm in B & D, and 50 µm in C. Blue arrows: Wolffian ducts; Magenta arrows: Müllerian ducts; White asterisk: ovary.