Catenin Expression in the Müllerian Duct Mesenchyme

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Catenin Expression in the Müllerian Duct Mesenchyme Focal Müllerian duct retention in male mice with constitutively activated β-catenin expression in the Müllerian duct mesenchyme Pradeep S. Tanwara, LiHua Zhanga, Yoshihiro Tanakaa, Makoto M. Taketob, Patricia K. Donahoec,1, and Jose M. Teixeiraa,1 aVincent Center For Reproductive Biology, Department of Obstetrics, Gynecology and Reproductive Biology, and cPediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; and bDepartment of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan Contributed by Patricia K. Donahoe, August 4, 2010 (sent for review April 23, 2010) Müllerian-inhibiting substance (MIS), which is produced by fetal β-catenin stabilization or a Wnt/Ca2+ pathway (12–14). The sta- Sertoli cells shortly after commitment of the bipotential gonads to bility of free β-catenin in the cytoplasm is regulated by a de- testicular differentiation, causes Müllerian duct (MD) regression. In struction complex formed by axin, adenomatosis polyposis the fetal female gonads, MIS is not expressed and the MDs will complex (APC), casein kinase 1, and glycogen synthase kinase 3-β differentiate into the internal female reproductive tract. We have (GSK3β), which binds and phosphorylates β-catenin, making it investigated whether dysregulated β-catenin activity affects MD re- a target for ubiquitination and degradation. During canonical Wnt gression by expressing a constitutively activated nuclear form of signaling, β-catenin escapes this complex and translocates to the β-catenin in the MD mesenchyme. We show that constitutively acti- nucleus to activate the transcription of various targeted genes in vated (CA) β-catenin causes focal retention of MD tissue in the epi- concert with members of the TCF/LEF family of transcription didymides and vasa deferentia. In adult mutant mice, the retained factors. Nuclear β-catenin expression in the MD mesenchyme has MD tissues express α-smooth muscle actin and desmin, which are been suggested as a mediator of MIS-induced MD regression (15). markers for uterine differentiation. MD retention inhibited the fold- We have previously shown that expression of a tissue-specific, ing complexity of the developing epididymides and usually led to constitutively activated (CA) form of β-catenin (16) driven by Cre BIOLOGY obstructive azoospermia by spermatoceles. The MDs of urogenital in the Amhr2 locus (AMHR2-Cre) did not result in MD regression DEVELOPMENTAL ridges from mutant female embryos showed less regression with in female mice (17), suggesting that β-catenin is not sufficient added MIS in organ culture compared with control MDs when ana- for regression. lyzed by whole mount in situ hybridization for Wnt7a as a marker We have previously reported that expression of CA β-catenin for the MD epithelium. CA β-catenin did not appear to affect expres- restricted to the uterine stroma and myometrium leads to myo- sion of either MIS in the embryonic testes or its type II receptor metrial smooth muscle cell hyperplasia and development of mes- (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nu- enchymal tumors in murine uteri (17). We and others have also clear accumulation, suggesting that dysregulated β-catenin must in- shown that CA β-catenin causes sterility in males by inhibiting hibit MD regression independently of MIS signaling. These studies postnatal Sertoli maturation and spermatogenesis (18–20). Given suggest that dysregulated Wnt/β-catenin signaling in the MD mes- the requirement for Wnt signaling in MD development (21) and enchyme might also be a contributing factor in persistent Müllerian the relative paucity of reports linking Wnt signaling and Wolffian duct syndrome, a form of male pseudohermaphroditism, and devel- duct (WD) differentiation, we examined whether dysregulated opment of spermatoceles. β-catenin activity in the adjacent MD mesenchyme affected normal WD development into the epididymis, vas deferens, and seminal anti-Müllerian hormone | epididymis | Mullerian inhibiting substance type II vesicles (22, 23). We show that CA β-catenin expression in the MD receptor (MISRII or MISR2) | spermatocele | epididymis mesenchyme results in focal MD retention and adjacent WD ob- struction, which leads to azoospermia and infertility secondary to hortly after commitment of the bipotential embryonic mam- seminiferous tubule degeneration and defective spermatogenesis Smalian gonadal ridge to male development, Sertoli cells dif- that develops later in the adult (18, 19). ferentiate in the testes and produce Müllerian-inhibiting substance [MIS; also known as anti-Müllerian hormone (AMH)], a TGFβ Results β family member that causes Müllerian duct (MD) regression in To investigate whether dysregulated CA -catenin expression in males. MIS is not produced by the embryonic female gonads; thus, the MD mesenchyme affects differentiation of the adjacent WD, β-catenin the MDs differentiate into the oviduct, uterus, cervix, and upper we generated mutant mice that lack exon 3 of the gene Ctnnb1fl(ex3) portion of the vagina (1, 2). Persistent Müllerian duct syndrome ( ), which would otherwise contain the serine/threo- β (PMDS) is a form of human male pseudohermaphroditism that is nine residues normally phosphorylated by GSK3 before ubiq- the result of retained female reproductive tract tissue. Homozy- uitination (16), by mating with mice that express Cre recombinase Amhr2 fi β gous deletion or knockdown of either MIS (3); its type I receptors, in the locus (4). Con rmation of nuclear -catenin ex- pression in the MD mesenchyme between the WD and MD of both BMPR1A (Alk3) (4) and ACVR1 (Alk2) (5, 6); or type II receptor, Δ(ex3) male and female urogenital ridges of Amhr2-Cre;Ctnnb1 mice AMHR2 (also known as MISRII) (7), in mice leads to retention of Ctnnb1fl(ex3) the MDs, and mutations in either MIS or MIS receptors are is shown in Fig. 1. In the urogenital ridges of control thought to account for 85% of patients with PMDS (8). Proper MD differentiation in females requires Wnt signaling. For example, the uteri of mice with a deletion of Wnt7a are de- Author contributions: P.S.T. and J.M.T. designed research; P.S.T., L.Z., and Y.T. performed fi research; M.M.T. and P.K.D. contributed new reagents/analytic tools; P.S.T., M.M.T., P.K.D., cient in myometrial tissue, endometrial glands, and oviductal and J.M.T. analyzed data; and P.S.T., P.K.D., and J.M.T. wrote the paper. coiling (9, 10), and mice lacking Wnt5a have shortened uterine The authors declare no conflict of interest. horns and defective development of cervices and vaginas (11). 1To whom correspondence may be addressed. E-mail: [email protected] or Wnts bind to their respective cell surface frizzled or low density [email protected]. lipoprotein-related protein (LRP) receptors and signal through This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. two intracellular pathways, a canonical pathway induced by 1073/pnas.1011606107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011606107 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 mice, we observed membranous expression of β-catenin in the AB MD epithelium at embryonic day (E) 14.5 of both females (Fig. 1 A and C) and males (Fig. 1 E and G). In ridges from Amhr2-Cre; Δ(ex3) Ctnnb1 mice, membranous β-catenin continued to be ex- pressed in the MD epithelium and strong nuclear β-catenin ex- pression was observed the MD mesenchyme (Fig. 1 B, D, F,andH). By P1, the MD has completely regressed in control male embryos (Fig. 2 A and B), but MD remnants are still observed in the epi- Δ(ex3) didymides and vasa deferentia of Amhr2-Cre;Ctnnb1 males (Fig. 2 C and D), which persisted through adulthood (Fig. 2 E and F C D and Fig. S1). Whereas membranous β-catenin expression was observed in epididymal epithelia, predominantly nuclear β-catenin was ob- served in the cells surrounding the retained MD-derived tissue of Δ( Amhr2-Cre;Ctnnb1 ex3) postnatal mice (Fig. 3 A and B). We also observed elevated expression of TCF1, LEF1, and cyclinD1, which have been shown to be up-regulated by Wnt/β-catenin signaling (24–28), in the P1 remnants of MD mesenchyme in the mutants E F compared with the normal surrounding tissue (Fig. S2), indicating that the nuclear β-catenin we observed was functionally active. The retained MD mesenchyme differentiates into an enlarged, simple tube-like structure resembling a uterus that expresses α-smooth muscle actin (αSMA) and desmin (Fig. 3 C and D), which are in- dicative of myometrium and endometrial stroma, respectively, but Fig. 2. Focal inhibition of Müllerian duct regression in mice with constitu- AB tive activated β-catenin in the MD mesenchyme. Gross analyses of male re- productive tracts of a control Amhr2-Cre/+;YFP mouse (A and B) show complete MD regression by P1 and that of an Amhr2-Cre/+;Ctnnb1Δ(ex3)/+; YFP mouse show bilateral focal retention (C and D, indicated with arrow- heads). Müllerian duct tissue is visible along side the epididymides of PND20 mutant mice (E and F, indicated with arrowheads) but not in control epi- didymides. T, testis; E, epididymis; V, vasa deferentia; Ex3, Amhr2-Cre/+; Ctnnb1Δ(ex3)/+;YFP; C, control. C D without endometrial glands and luminal folds, and is similarly observed in mice homozygously deleted for either MIS or one of its receptors (3, 4). Histological analyses showed that the remaining Δ(ex3) MD-derived tissue in Amhr2-Cre;Ctnnb1 inhibited coiling and integrity of the ducts in the caput and corpus regions of the epi- didymides (Fig. 3 E–H). Analysis of cytokeratin expression also showed that the integrity of the pseudostratified columnar ductal epithelium in the cauda epididymides was compromised near the EF retained MD tissue in mutant mice (Fig.
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