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Supplementary Table 1 from Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Misregulation of the Wnt/β-catenin signaling pathway is a hallmark of several forms of cancer. Components of the Wnt/β-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 β-catenin expression by immunohistochemistry. Unlike granulosa cells of normal ovaries, most (15 of 24) GCT samples showed nuclear localization of β-catenin, suggesting that activation of the Wnt/β-catenin pathway plays a role in the etiology of GCT. To confirm this hypothesis, Catnbflox(ex3)/+; Amhr2cre/+ mice that express a dominant stable β-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. Catnbflox(ex3)/+; Amhr2cre/+ 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 Catnbflox(ex3)/+; Amhr2cre/+ 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/β-catenin signaling and GCT development and provide a novel model system for the study of GCT biology.

Supplementary Figure Legend from Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Supplementary Figure 1 from Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

International audience; EXOSC10 is a catalytic subunit of the nuclear RNA exosome, and possesses a 3’-5’ exoribonuclease activity. The enzyme processes and degrades different classes of RNAs. To delineate the role of EXOSC10 during oocyte growth, specific Exosc10 inactivation was performed in oocytes from the primordial follicle stage onward using the Gdf9-iCre; Exosc10 (f/-) mouse model (Exosc10 (cKO(Gdf9))). Exosc10 (cKO(Gdf9)) female mice are infertile. The onset of puberty and the estrus cycle in mutants are initially normal and ovaries contain all follicle classes. By the age of eight weeks, vaginal smears reveal irregular estrus cycles and mutant ovaries are completely depleted of follicles. Mutant oocytes retrieved from the oviduct are degenerated, and occasionally show an enlarged polar body, which may reflect a defective first meiotic division. Under fertilization conditions, the mutant oocytes do not enter into an embryonic development process. Furthermore, we conducted a comparative proteome analysis of wild type and Exosc10 knockout mouse ovaries, and identified EXOSC10-dependent proteins involved in many biological processes, such as meiotic cell cycle progression and oocyte maturation. Our results unambiguously demonstrate an essential role for EXOSC10 in oogenesis and may serve as a model for primary ovarian insufficiency in humans. Data are available via ProteomeXchange with identifier PXD039417.

Cancer/Testis (CT) genes are induced in germ cells, repressed in somatic cells, and derepressed in somatic tumors, where these genes can contribute to cancer progression. CT gene identification requires data obtained using standardized protocols and technologies. This is a challenge because data for germ cells, gonads, normal somatic tissues, and a wide range of cancer samples stem from multiple sources and were generated over substantial periods of time. We carried out a GeneChip‐based RNA profiling analysis using our own data for testis and enriched germ cells, data for somatic cancers from the Expression Project for Oncology, and data for normal somatic tissues from the Gene Omnibus Repository. We identified 478 candidate loci that include known CT genes, numerous genes associated with oncogenic processes, and novel candidates that are not referenced in the Cancer/Testis Database (www.cta.lncc.br). We complemented RNA expression data at the protein level for SPESP1, GALNTL5, PDCL2, and C11orf42 using cancer tissue microarrays covering malignant tumors of breast, uterus, thyroid, and kidney, as well as published RNA profiling and immunohistochemical data provided by the Human Protein Atlas (www.proteinatlas.org). We report that combined RNA/tissue profiling identifies novel CT genes that may be of clinical interest as therapeutical targets or biomarkers. Our findings also highlight the challenges of detecting truly germ cell‐specific mRNAs and the proteins they encode in highly heterogenous testicular, somatic, and tumor tissues., Cancer/Testis (CT) genes are induced in germ cells, repressed in somatic cells, and derepressed in somatic tumors where they can contribute to cancer progression. We report that combined RNA/tissue profiling identifies novel CT genes with potential oncogenic functions and highlight the challenges of detecting germ cell‐specific mRNAs and the proteins they encode in highly heterogenous testicular, somatic, and tumor tissues.

The conserved 3’-5’ exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10−/− mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the protein’s role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10−/− mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

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International audience; Mullerian 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-beta) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Mullerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Mullerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Mullerian 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 alpha. As previously described in a beta-catenin conditional mutant mouse model, beta-catenin contributes to Mullerian duct regression. In our mutant male embryos, it appears that beta-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/beta-catenin signalling leading to the partial persistence of Mullerian duct

International audience; Plasma anti-Müllerian hormone (AMH) concentrations have been recently found to be predictive of the number of embryos recovered after FSH superovulatory treatment in the cow. However, the sensitivity of the Active Müllerian-inhibiting substance/AMH ELISA (ref. 10–14400; DSL-Beckman-Coulter) used to make these measurements in bovine plasma samples is low because it was developed to measure human AMH levels. To overcome this limitation, we developed an immunoassay specific for the bovine (B), ovine (O), and caprine (C) species, the bovine-ovine-caprine (BOC) ELISA. For this purpose, we produced recombinant bovine AMH for standardization, and we used monoclonal antibodies raised against bovine AMH, previously prepared by our laboratory. We evaluated the precision, accuracy, specificity, limit of detection, and functional sensitivity of the assay. The intra-assay coefficient of variation ranged between 3.4% and 11.3% for AMH concentrations between 23.68 and 1.74 ng/mL, and the interassay coefficient of variation ranged between 4.8% and 20.5% for concentrations between 25.53 and 1.42 ng/mL, respectively. The assay displayed a good linearity, had a detection limit of 0.4 ng/mL and a functional sensitivity of 1.4 ng/mL. It also cross-reacted with ovine and caprine AMHs. Both the mean and median AMH levels measured in 40 cow plasma samples using the BOC ELISA were approximately 44 fold higher than the mean and median AMH levels measured with the Active Müllerian-inhibiting substance/AMH ELISA. Moreover, a higher correlation was observed between the average number of embryos recovered from each cow after superovulatory treatment and AMH concentrations measured with the BOC ELISA. This BOC ELISA provides a very efficient tool for evaluating the ovarian follicular reserve of cows and predicting their embryo production capacity.

EXOSC10 is a catalytic subunit of the exosome that processes biologically active transcripts, degrades aberrant mRNAs and targets certain long non-coding RNAs (lncRNAs). The yeast orthologue Rrp6 is required for efficient growth and gametogenesis, and becomes unstable during meiosis. However, nothing is known about the localization, stability and function of EXOSC10 in the rodent male germline. We detect the protein in nucleoli and the cytoplasm of mitotic and meiotic germ cells, and find that it transiently associates with the XY body, a structure targeted by meiotic sex chromosome inactivation (MSCI). Finally, EXOSC10 becomes unstable at later stages of gamete development. To determine Exosc10’s meiotic function, we inactivated the gene specifically in male germ cells using cre recombinase controlled by Stra8 or Ddx4/Vasa promoters. Mutant mice have small testes, show impaired germ cell differentiation and are subfertile. Our results demonstrate that EXOSC10 is post-translationally regulated in germ cells, associate the protein with epigenetic chromosome silencing, and reveal its essential role in germ cell growth and development.

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.

The balance between tissue-type plasminogen activator (tPA) and one of its inhibitors, neuroserpin, has crucial roles in the central nervous system, including the control of neuronal migration, neuronal plasticity and neuronal death. In the present study, we demonstrate that the activation of the transforming growth factor-β (TGFβ)-related BMPR-IB (also known as BMPR1B and Alk6)- and Smad5-dependent signalling pathways controls neuroserpin transcription. Accordingly, we demonstrate for the first time that anti-Mullerian hormone (AMH), a member of the TGFβ family, promotes the expression of neuroserpin in cultured neurons but not in astrocytes. The relevance of these findings is confirmed by the presence of both AMH and AMH type-II receptor (AMHR-II) in brain tissues, and is supported by the observation of reduced levels of neuroserpin in the brain of AMHR-II-deficient mice. Interestingly, as previously demonstrated for neuroserpin, AMH protects neurons against N-methyl-D-aspartate (NMDA)-mediated excitotoxicity both in vitro and in vivo. This study demonstrates the existence of an AMH-dependent signalling pathway in the brain leading to an overexpression of the serine-protease inhibitor, neuroserpin, and neuronal survival.

Genome-wide RNA profiling studies have identified hundreds of transcripts that are highly expressed in mammalian male germ cells, including many that are undetectable in somatic control tissues. Among them, genes important for spermatogenesis are significantly enriched. Information about mRNAs and their cognate proteins facilitates the identification of novel conserved target genes for functional studies in the mouse. By inspecting genome-wide RNA profiling data, we manually selected 81 genes for which RNA is detected almost exclusively in the human male germline and, in most cases, in rodent testicular germ cells. We observed corresponding mRNA/protein patterns in 43 cases using immunohistochemical data from the Human Protein Atlas and large-scale human protein profiling data obtained via mass spectroscopy. Protein network information enabled us to establish an interaction map of 38 proteins that points to potentially important testicular roles for some of them. We further characterized six candidate genes at the protein level in the mouse. We conclude that conserved genes induced in testis tend to show similar mRNA/protein expression patterns across species. Specifically, our results suggest roles during embryogenesis and adult spermatogenesis for Foxr1 and Sox30 and during spermiogenesis and fertility for Fam71b, 1700019N19Rik, Hmgb4, and Zfp597.

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor α and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.

International audience; Anti-Mullerian hormone (AMH) is a member of the TGF-b superfamily secreted by the gonads of both sexes. This hormone is primarily known for its role in the regression of the Mullerian ducts in male fetuses. In females, AMH is expressed in granulosa cells of developing follicles. Like other members of the TGF-b superfamily, AMH transduces its signal through two transmembrane serine/threonine kinase receptors including a well characterized type II receptor, AMHR-II. The complete signalling pathway of AMH involving Smads proteins and the type I receptor is well known in the Mullerian duct and in Sertoli and Leydig cells but not in granulosa cells. In addition, few AMH target genes have been identified in these cells. Finally, while several co-receptors have been reported for members of the TGF-b superfamily, none have been described for AMH. Here, we have shown that none of the Bone Morphogenetic Proteins (BMPs) co-receptors, Repulsive guidance molecules (RGMs), were essential for AMH signalling. We also demonstrated that the main Smad proteins used by AMH in granulosa cells were Smad 1 and Smad 5. Like for the other AMH target cells, the most important type I receptor for AMH in these cells was BMPR-IA. Finally, we have identified a new AMH target gene, Id3, which could be involved in the effects of AMH on the differentiation of granulosa cells and its other target cells.

International audience; 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.

TGF-β family ligands are translated as prepropeptide precursors and are processed into mature C-terminal dimers that signal by assembling a serine/threonine kinase receptor complex containing type I and II components. Many TGF-β ligands are secreted in a latent form that cannot bind their receptor, due to the pro-region remaining associated with the mature ligand in a noncovalent complex after proteolytic cleavage. Here we show that anti-Müllerian hormone (AMH), a TGF-β family ligand involved in reproductive development, must be cleaved to bind its type II receptor (AMHRII), but dissociation of the pro-region from the mature C-terminal dimer is not required for this initial interaction. We provide direct evidence for this interaction by showing that the noncovalent complex binds to a soluble form of AMHRII in an ELISA format and to AMHRII immobilized on Sepharose. Binding of the noncovalent complex to Sepharose-coupled AMHRII induces dissociation of the pro-region from the mature C-terminal dimer, whereas no dissociation occurs after binding to immobilized AMH antibodies. The pro-region cannot be detected after binding of the AMH noncovalent complex to AMHRII expressed on COS cells, indicating that pro-region dissociation may occur as a natural consequence of receptor engagement on cells. Moreover, the mature C-terminal dimer is more active than the noncovalent complex in stimulating Sma- and Mad-related protein activation, suggesting that pro-region dissociation contributes to the assembly of the active receptor complex. AMH thus exemplifies a new mechanism for receptor engagement in which interaction with the type II receptor promotes pro-region dissociation to generate mature ligand.

In the polycystic ovary syndrome (PCOS), in addition to intrinsic thecal dysregulation leading to hyperandrogenism, a granulosa cell (GC) dysregulation may occur. Expression of anti-Müllerian hormone (AMH), FSH receptor (FSHR) and androgen receptor (AR) are suspected to be altered in PCOS GCs.The aim of this prospective study was to analyze the expression of these genes at the last stages of follicular maturation in GCs from 17 patients with PCOS and 15 controls undergoing controlled ovarian hyperstimulation during a cycle with in vitro fertilization.On the day of oocyte retrieval, follicular fluids were collected from small follicles (SF; 8-13 mm) and large follicles (17-22 mm) in separate tubes. Total RNAs and proteins were extracted from GCs. Reverse transcription was performed and quantification of gene expression levels was achieved by real-time quantitative PCR.AMH and FSHR mRNA levels were significantly higher in PCOS than in controls in GCs from both SF and large follicles. Likewise, AR and AMH receptor II mRNA levels in GCs from SF were significantly higher in PCOS compared with controls. In both PCOS patients and controls, AMH and AR mRNA levels correlated strongly, positively, and independently to FSHR mRNA levels.Using quantitative RT-PCR, AMH, AMH receptor II, FSHR, and AR genes were shown to be overexpressed by GCs from stimulated follicles of women with PCOS undergoing controlled ovarian hyperstimulation. This could be the sign of a maturation defect or may reflect hyperandrogenism.

The Mullerian ducts are composed of an epithelium and surrounding mesenchyme that have the potential to differentiate into female reproductive organs, including the oviducts, uterus and upper vagina. In eutherian mammals, Mullerian inhibiting substance/anti-Mullerian hormone (MIS/AMH) secreted by the fetal testis causes the regression of the Mullerian ducts to prevent the differentiation of female reproductive organs in males. MIS signalling in the Mullerian duct is mediated by the MIS type II receptor (MISRII) that is expressed in the mesenchyme surrounding the epithelium. MIS signalling alters the Mullerian duct mesenchyme, leading to the elimination of the ductal epithelium. Loss of MIS signalling, by mutation of MIS or MISRII, leads to the differentiation of female reproductive organs in males that can cause cryptorchidism and infertility. We have exploited the mouse MisrII locus to express heterologous genes in the cellular target of MIS signalling, the Mullerian duct mesenchyme. This approach can be used with conditional genetic strategies to identify factors that are required for the regression of the female genital duct system.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Mullerian 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 Mullerian 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-Mullerian hormone, which activates the regression of the Mullerian duct. Anti-Mullerian 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-Mullerian hormone receptor 2 (AMHR2), in the Mullerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Mullerian 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-Mullerian hormone signal required for Mullerian duct regression. Loss of these genes in the Mullerian duct mesenchyme results in male infertility due to retention of Mullerian duct derivatives in an otherwise virilized male.

The Mullerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Mullerian 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 Mullerian 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 Mullerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Mullerian 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 Mullerian duct regression. This pattern was similar to that observed using a Mullerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Mullerian duct mesoepithelium and Amhr2-expressing mesenchyme. Mol. Reprod. Dev. 75: 1154–1162, 2008. © 2008 Wiley-Liss, Inc.

The transforming growth factor beta (TGF beta) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGF beta 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 TGF beta 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. ispartof: Molecular and cellular biology vol:28 issue:1 pages:248-257 ispartof: location:United States status: published

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.

Misregulation of the Wnt/β-catenin signaling pathway is a hallmark of several forms of cancer. Components of the Wnt/β-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 β-catenin expression by immunohistochemistry. Unlike granulosa cells of normal ovaries, most (15 of 24) GCT samples showed nuclear localization of β-catenin, suggesting that activation of the Wnt/β-catenin pathway plays a role in the etiology of GCT. To confirm this hypothesis, Catnbflox(ex3)/+; Amhr2cre/+ mice that express a dominant stable β-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. Catnbflox(ex3)/+; Amhr2cre/+ 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 Catnbflox(ex3)/+; Amhr2cre/+ 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/β-catenin signaling and GCT development and provide a novel model system for the study of GCT biology.

International audience; Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-beta family responsible for regression of Müllerian ducts during male sexual differentiation and for regulation of gonadal steroidogenesis. AMH is also a gonadal tumor suppressor which mediates its effects through a specific type II receptor and the bone morphogenetic protein (BMP)-specific Smad proteins, suggesting that AMH and BMPs could also share type I receptors, namely activin-like kinases (ALKs)2, 3 or 6. However, attempts to identify a unique AMH type I receptor among them were unsuccessful. Here, using kinase-deficient type I receptors and small interfering RNA technology, we demonstrate that, in an AMH Sertoli target cell line, ALK3 mediates AMH effects on both Smad1 activation and P450 side-chain cleavage enzyme. In addition, transfecting a combination of normal and kinase-deficient receptors, we show that ALK2 can compensate for the absence of ALK3 and probably acts in synergy with ALK3 at high concentrations of AMH to activate Smad1, whereas ALK6 has a competitive inhibitory effect. These results are a first step in understanding how AMH transduces its effects in immature Sertoli cells.

Follistatin plays an important role in female physiology by regulating FSH levels through blocking activin actions. Failure to regulate FSH has been implicated as a potential cause of premature ovarian failure. Premature ovarian failure is characterized by amenorrhea, infertility, and elevated gonadotropin levels in women under the age of 40. Because follistatin is essential for postnatal viability, we designed a cre/loxP conditional knockout system to render the follistatin gene null specifically in the granulosa cells of the postnatal ovary using Amhr2cre transgenic mice. The follistatin conditional knockout females develop fertility defects, including reduced litter number and litter sizes and, in the most severe case, infertility. Reduced numbers of ovarian follicles, ovulation and fertilization defects, elevated levels of serum FSH and LH, and reduced levels of testosterone were observed in these mice. These findings demonstrate that compromising granulosa cell follistatin function leads to findings similar to those characterized in premature ovarian failure. Follistatin conditional knockouts may therefore be a useful model with which to further study this human syndrome. These studies are the first report of a granulosa cell-specific deletion of a gene in the postnatal ovary and have important implications for future endeavors to generate ovary-specific knockout mouse models.

Anti-Mullerian hormone (AMH)/Mullerian-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 Mullerian 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 Mullerian duct, resulting in males with a uterus. Thus, Bmpr1a plays an essential role in the process of Mullerian 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 Mullerian duct had no uterus. These results suggest that when AMH is overexpressed, other TGF-beta family type I receptors can potentially transduce AMH signals.

Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation1. In males, the fetal testis produces the transforming growth factor β (TGF-β) family member anti-Mullerian hormone (Amh, also known as Mullerian-inhibiting substance (Mis)), which causes regression of the Mullerian ducts, the primordia of the oviducts, uterus and upper vagina2. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium3,4,5,6,7. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri8,9,10. The type I receptor and Smad proteins that are required in vivo for Mullerian duct regression have not yet been identified11,12,13,14. 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 Mullerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Mullerian 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.

The Müllerian ducts are composed of an epithelium and surrounding mesenchyme that have the potential to differentiate into female reproductive organs, including the oviducts, uterus and upper vagina. In eutherian mammals, Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) secreted by the fetal testis causes the regression of the Müllerian ducts to prevent the differentiation of female reproductive organs in males. MIS signalling in the Müllerian duct is mediated by the MIS type II receptor (MISRII) that is expressed in the mesenchyme surrounding the epithelium. MIS signalling alters the Müllerian duct mesenchyme, leading to the elimination of the ductal epithelium. Loss of MIS signalling, by mutation of MIS or MISRII, leads to the differentiation of female reproductive organs in males that can cause cryptorchidism and infertility. We have exploited the mouse MisrII locus to express heterologous genes in the cellular target of MIS signalling, the Müllerian duct mesenchyme. This approach can be used with conditional genetic strategies to identify factors that are required for the regression of the female genital duct system.

Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.

A region of homology, containing the contiguous SF3A2 (formerly called SAP62) and AMH genes, exists between human chromosome 19 (HSA19p) and mouse chromosome 10 (MMU10). In a previous study it was shown that SF3A2/Sf3a2 is very highly conserved between the two species and that AMH/Amh is somewhat less conserved although both human and mouse genes encode a protein (AMH) playing the same critical role during early male sex differentiation. The close association between SF3a2/Sf3a2 and AMH/Amh was thought to maintain open chromatin in the AMH/Amh promoter region, thus facilitating the necessary precise timing of AMH/Amh expression following that of SRY/Sry at the onset of testis differentiation. Further investigation of DNA upstream of Amh has revealed that there is another gene, in close association (about 400 bp) with Sf3a2, which has significant similarities to the N-terminus of a known guanine nucleotide releasing protein (GNRP) and consequently is provisionally named GNRPx/Gnrpx. The Gnrpx-Sf3a2-Amh (GSA) locus of the mouse (MMU10) is conserved in the human (HSA19p). Mapping the Sf3a2 transcription start site eventually led us to locate and characterize its promoter. We found that Sf3a2 and Gnrpx share a bi-directional promoter, with the latter being transcribed in an antisense direction. It has now been shown by RT-PCR analysis that both Sf3a2 and Gnrpx are widely expressed and therefore are likely to be ‘housekeeping’ genes. GNRPx/Gnrpx messenger RNA codes for a C-terminally truncated protein (149/164 aa), which contains an as yet uncharacterized domain common to GNRPs (and related proteins) and which may therefore act as a specific antagonist of a complete GNRP protein (>1200 aa) involved in the regulation of the GTPase (G-protein/Ras) cycle.