Focal Mullerian duct retention in male mice with constitutively activated beta-catenin expression in the Mullerian duct mesenchyme

Abstract

Müllerian-inhibiting substance (MIS), which is produced by fetal Sertoli cells shortly after commitment of the bipotential gonads to testicular differentiation, causes Müllerian duct (MD) regression. In the fetal female gonads, MIS is not expressed and the MDs will differentiate into the internal female reproductive tract. We have investigated whether dysregulated β-catenin activity affects MD regression by expressing a constitutively activated nuclear form of β-catenin in the MD mesenchyme. We show that constitutively activated (CA) β-catenin causes focal retention of MD tissue in the epididymides and vasa deferentia. In adult mutant mice, the retained MD tissues express α-smooth muscle actin and desmin, which are markers for uterine differentiation. MD retention inhibited the folding complexity of the developing epididymides and usually led to obstructive azoospermia by spermatoceles. The MDs of urogenital ridges from mutant female embryos showed less regression with added MIS in organ culture compared with control MDs when analyzed by whole mount in situ hybridization for Wnt7a as a marker for the MD epithelium. CA β-catenin did not appear to affect expression of either MIS in the embryonic testes or its type II receptor (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nuclear accumulation, suggesting that dysregulated β-catenin must inhibit MD regression independently of MIS signaling. These studies suggest that dysregulated Wnt/β-catenin signaling in the MD mesenchyme might also be a contributing factor in persistent Müllerian duct syndrome, a form of male pseudohermaphroditism, and development of spermatoceles.

Fig. 1.

Nuclear β-catenin expression in the MD mesenchyme. Membranous expression of β-catenin is observed by immunofluorescence (green) in E14.5 male and female urogenital ridges of control Amhr2-Cre/+ (A, C, E, G) in the epithelial cells but is largely nuclear in the MD mesenchyme of Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+ (B, D, F, H) embryos. DAPI staining is shown in blue to mark nuclei. Gonocytes cells are shown by mvh immunofluorescence (red). O, ovary; T, testis; MDM, Müllerian duct mesenchyme; MDE, Müllerian duct epithelium; WD, Wolffian duct. (Scale bars, 50 μm.)

Fig. 2.

Focal inhibition of Müllerian duct regression in mice with constitutive activated β-catenin in the MD mesenchyme. Gross analyses of male reproductive tracts of a control Amhr2-Cre/+;YFP mouse (A and B) show complete MD regression by P1 and that of an Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+;YFP mouse show bilateral focal retention (C and D, indicated with arrowheads). Müllerian duct tissue is visible along side the epididymides of PND20 mutant mice (E and F, indicated with arrowheads) but not in control epididymides. T, testis; E, epididymis; V, vasa deferentia; Ex3, Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+;YFP; C, control.

Fig. 3.

Müllerian duct remnants in Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+ male mice adversely affect the development and function of the epididymides and vasa deferentia. Only membranous β-catenin immunofluorescence was observed in the control Ctnnb1^Δ(ex3)/+ and mutant Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+ epididymal ductal epithelia of P1 mice (A and B). However, strong nuclear accumulation of the β-catenin was observed in the uterine mesenchyme of the mutant animals (B). Desmin and αSMA were limited to the stroma and periductal cells, respectively, of the control and mutant epididymides (C and D) and to the inner and outer areas of the retained MD tissue (D). DAPI staining is shown in blue to mark nuclei. Histology of the caput epididymus shows fewer ducts in the areas with retained MD tissue of mutant mice compared with the controls (E and F). The number and quality of the ducts (evaluated by histology) appeared normal in the corpus epididymus near the retained MD tissue (G and H). However, immunostaining for keratins showed that the integrity of the ductal epithelium was compromised in the mutants (I and J). In vasa deferentia, H&E staining showed that the presence of the vestigial uterine tube markedly diminishes the luminal folding in the neighboring vas deferens in the mutants compared with normal vas deferens in control animals (K and L). (Scale bars, 50 μm.) U, vestigial uterine remnant; E, epididymis; V, vas deferens.

Fig. 4.

Spermatocele formation in mutant mice. The epididymides of adult Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+ mice were analyzed by H&E. Obstructive azoospermia were observed upstream of retained fibrotic (F) MD tissue (A). (B) An epididymis from another mutant with a large spermatocele (S). The boxes show the areas enlarged in C and D, which show the absence of sperm in the duct downstream of the spermatocele and the presence of sperm in the duct upstream, respectively. (Scale bar = 50 μm.)

Fig. 5.

Müllerian duct regression in mutant urogenital ridges is inhibited in organ culture. Urogenital ridges from E13.5 control and mutant female fetuses were isolated, incubated for 48 h with 5 μg/mL MIS, and subjected to whole mount in situ hybridization with Wnt7a riboprobe detected by BM purple. (A) There is no Wnt7a mRNA expression in control female urogenital ridge with MIS, indicating complete regression of MD epithelium. (Inset) Wnt7a expression in a control female urogenital without added MIS (indicated with a white arrowhead). (B) Complete MD retention (indicated with a white arrowhead) with an Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+ female urogenital ridge similarly incubated with MIS. In situ hybridization for Amhr2 mRNA in control (C) and Amhr2-Cre/+;Ctnnb1^Δ(ex3)/+ (D) E16.5 male reproductive tract shows expression in the retained MD tissue in the mutant (indicated with black arrowheads in the epididymis and a black arrow in the vas deferens). pSmad1/5/8 expression was detected by immunofluorescence in control (E) and mutant (F) E14.5 male urogenital ridges. DAPI staining in blue indicates nuclei. O, ovary; T, testis; E, epididymis; MDM, Müllerian duct mesenchyme; MDE, Müllerian duct epithelium; WD, Wolffian duct. (Scale bars, 50 μm.)