Soazik P. Jamin

Requirement of Bmpr1a for Müllerian duct regression during male sexual development.

Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation. In males, the fetal testis produces the transforming growth factor β (TGF-β) family member anti-Müllerian hormone (Amh, also known as Müllerian-inhibiting substance (Mis)), which causes regression of the Müllerian ducts, the primordia of the oviducts, uterus and upper vagina. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri. The type I receptor and Smad proteins that are required in vivo for Müllerian duct regression have not yet been identified. Here we show that targeted disruption of the widely expressed type I bone morphogenetic protein (BMP) receptor Bmpr1a (also known as Alk3) in the mesenchymal cells of the Müllerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Müllerian ducts. Because Bmpr1a is evolutionarily conserved, these findings indicate that a component of the BMP signaling pathway has been co-opted during evolution for male sexual development in amniotes.

Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice

ABSTRACT The transforming growth factor β (TGFβ) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGFβ function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGFβ pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.

Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Misregulation of the Wnt/beta-catenin signaling pathway is a hallmark of several forms of cancer. Components of the Wnt/beta-catenin pathway are expressed in ovarian granulosa cells; nevertheless, its potential involvement in granulosa cell tumorigenesis has not been examined. To this end, human (n = 6) and equine (n = 18) granulosa cell tumors (GCT) were analyzed for beta-catenin expression by immunohistochemistry. Unlike granulosa cells of normal ovaries, most (15 of 24) GCT samples showed nuclear localization of beta-catenin, suggesting that activation of the Wnt/beta-catenin pathway plays a role in the etiology of GCT. To confirm this hypothesis, Catnb(flox(ex3)/+); Amhr2(cre/+) mice that express a dominant stable beta-catenin mutant in their granulosa cells were generated. These mice developed follicle-like structures containing disorganized, pleiomorphic granulosa by 6 weeks of age. Even in older mice, these follicle-like lesions grew no larger than the size of antral follicles and contained very few proliferating cells. Similar to corpora lutea, the lesions were highly vascularized, although they did not express the luteinization marker Cyp11a1. Catnb(flox(ex3)/+); Amhr2(cre/+) females were also found to be severely subfertile, and fewer corpora lutea were found to form in response to exogenous gonadotropin compared with control mice. In older mice, the ovarian lesions often evolved into GCT, indicating that they represent a pretumoral intermediate stage. The GCT in Catnb(flox(ex3)/+); Amhr2(cre/+) mice featured many histopathologic similarities to the human disease, and prevalence of tumor development attained 57% at 7.5 months of age. Together, these studies show a causal link between misregulated Wnt/beta-catenin signaling and GCT development and provide a novel model system for the study of GCT biology.

A mesenchymal perspective of müllerian duct differentiation and regression in Amhr2-lacZ mice

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding β‐galactosidase (β‐gal), into the AMHR‐II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2‐lacZ heterozygotes express β‐gal activity in an Amhr2‐specific pattern. In the gonads, β‐gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. β‐gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, β‐gal activity was restricted to one side of the ductal mesoepithelium, whereas in males β‐gal expression encircled the duct. β‐gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal β‐gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2‐expressing mesenchyme. Mol. Reprod. Dev. 75: 1154–1162, 2008.

Functional Redundancy of TGF-beta Family Type I Receptors and Receptor-Smads in Mediating Anti-Müllerian Hormone-Induced Müllerian Duct Regression in the Mouse

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Deletion of the orphan nuclear receptor COUP-TFII in uterus leads to placental deficiency

COUP-TFII (NR2F2), chicken ovalbumin upstream promoter–transcription factor II, is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. The Coup-tfII-null mutant mice die during the early embryonic development because of angiogenesis and heart defects. To analyze the physiological function of COUP-TFII during organogenesis, we used the cre/loxP system to conditionally inactivate COUP-TFII in the ovary and uterus. Homozygous adult female mutants with specific inactivation of the Coup-tfII gene in uterine stromal and smooth muscle cells have severely impaired placental formation, leading to miscarriage at days 10–12 of pregnancy. Deletion of the Coup-tfII gene resulted in an increase in trophoblast giant cell differentiation, a reduction of the spongiotrophoblast layer, and an absence of labyrinth formation causing an improper vascularization of the placenta. This study describes an important maternal role of COUP-TFII in regulating the placentation. The endometrial COUP-TFII might modulate the signaling between the uterus and the extraembryonic tissue for the proper formation of the placenta.

Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression

The mechanisms of granulosa cell tumor (GCT) development may involve the dysregulation of signaling pathways downstream of follicle-stimulating hormone, including the phosphoinosite-3 kinase (PI3K)/AKT pathway. To test this hypothesis, a genetically engineered mouse model was created to derepress the PI3K/AKT pathway in granulosa cells by conditional targeting of the PI3K antagonist gene Pten (Pten(flox/flox);Amhr2(cre/+)). The majority of Pten(flox/flox);Amhr2(cre/+) mice featured no ovarian anomalies, but occasionally ( approximately 7%) developed aggressive, anaplastic GCT with pulmonary metastases. The expression of the PI3K/AKT downstream effector FOXO1 was abrogated in Pten(flox/flox);Amhr2(cre/+) GCT, indicating a mechanism by which GCT cells may increase proliferation and evade apoptosis. To relate these findings to spontaneously occurring GCT, analyses of PTEN and phospho-AKT expression were performed on human and equine tumors. Although PTEN loss was not detected, many GCT (2/5 human, 7/17 equine) featured abnormal nuclear or perinuclear localization of phospho-AKT, suggestive of altered PI3K/AKT activity. As inappropriate activation of WNT/CTNNB1 signaling causes late-onset GCT development and cross talk between the PI3K/AKT and WNT/CTNNB1 pathways has been reported, we tested whether these pathways could synergize in GCT. Activation of both the PI3K/AKT and WNT/CTNNB1 pathways in the granulosa cells of a mouse model (Pten(flox/flox);Ctnnb1(flox(ex3)/+);Amhr2(cre/+)) resulted in the development of GCT similar to those observed in Pten(flox/flox);Amhr2(cre/+) mice, but with 100% penetrance, perinatal onset, extremely rapid growth and the ability to spread by seeding into the abdominal cavity. These data indicate a synergistic effect of dysregulated PI3K/AKT and WNT/CTNNB1 signaling in the development and progression of GCT and provide the first animal models for metastatic GCT.

β-Catenin is essential for Müllerian duct regression during male sexual differentiation

During male sexual differentiation, the transforming growth factor-β (TGF-β) signaling molecule anti-Müllerian hormone (AMH; also known as Müllerian inhibiting substance, MIS) is secreted by the fetal testes and induces regression of the Müllerian ducts, the primordia of the female reproductive tract organs. Currently, the molecular identity of downstream events regulated by the AMH signaling pathway remains unclear. We found that male-specific Wnt4 expression in mouse Müllerian duct mesenchyme depends upon AMH signaling, implicating the WNT pathway as a downstream mediator of Müllerian duct regression. Inactivation of β-catenin, a mediator of the canonical WNT pathway, did not affect AMH signaling activation in the Müllerian duct mesenchyme, but did block Müllerian duct regression. These data suggest that β-catenin mediates AMH signaling for Müllerian duct regression during male sexual differentiation.

Genetic studies of the AMH/MIS signaling pathway for Müllerian duct regression

Anti-Müllerian hormone (AMH)/Müllerian-inhibiting substance (MIS) is a member of the transforming growth factor-beta (TGF-beta) superfamily. Like other TGF-beta family members, AMH is likely to signal through two transmembrane serine/threonine kinase receptors. Whereas the AMH type II receptor has been clearly defined, only recently has there been evidence about the identity of the AMH type I receptor for Müllerian duct regression in vivo. We generated a new cre mouse line expressing the recombinase in AMH target cells. This line was then used to conditionally inactivate the Bmpr1a gene in the Müllerian duct, resulting in males with a uterus. Thus, Bmpr1a plays an essential role in the process of Müllerian duct regression. To investigate the role of Bmpr1a in granulosa cells, we took advantage of transgenic mice overexpressing human AMH. Surprisingly, these transgenic females that were also conditionally mutant for Bmpr1a in the Müllerian duct had no uterus. These results suggest that when AMH is overexpressed, other TGF-beta family type I receptors can potentially transduce AMH signals.

Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice.

Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.