FOXR1 belongs to the large conserved F-box family of DNA binding transcription factors that are involved in various developmental processes and diseases. The gene is important for embryogenesis in a fish model, brain development in mammals, and human FOXR1 was shown to possess oncogenic properties when it is abnormally expressed as a fusion gene. Earlier work on Foxr1 expression in mouse and human suggested roles for the protein in embryogenesis and sexual reproduction. We generated a mouse gene deletion model that revealed an embryonic lethal phenotype with partial penetrance and normal fertility in persistent homozygous male mutants. The results suggest that Foxr1 is functionally redundant in adult male gonads but needed for normal early embryo development and post-natal viability. We discuss our results in the context of publicly available human and rodent genomic and genetic data.

This study aims to explore the genetic anomalies (excluding aneuploidy) associated with Müllerian duct anomalies (MDA). This project is part of a large uterine transplantation (UTx) program, where women with MRKH (Mayer-Rokitansky-Küster-Hauser) syndrome are the primary candidates. The potential of the hereditary nature of MDA was evaluated to facilitate appropriate genetic counseling when applicable. We used chromosome microarray analysis (CMA) and exome sequencing (ES) to identify genetic variations that may contribute to the phenotype of MDA. A comprehensive description of the genital and extra-genital phenotype was established for each individual in whom a potentially relevant genetic variant has been identified. We analyzed data from 3 fetuses and 75 women with MDA. All individuals had CMA and 65 ES were analyzed. CMA identified 6 copy number variations (CNVs) of interest (diagnostic yield: 7.7

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

In female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus.

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.

A transgenic mouse approach using bacterial artificial chromosomes (BAC) was used to identify regulatory regions that direct Müllerian duct expression for Amhr2 and Osterix (Osx, also known as Sp7). Amhr2 encodes the receptor that mediates anti-Müllerian hormone (AMH) signaling for Müllerian duct regression in male embryos. Amhr2 is expressed in the Müllerian duct mesenchyme of both male and female embryos. A ∼147-kb BAC clone containing the Amhr2 locus was used to generate transgenic mice. The transgene was able to rescue the block in Müllerian duct regression of Amhr2-null males, suggesting that the BAC clone contains regulatory sequences active in male embryos. Osx is expressed in the developing skeleton of male and female embryos but is also an AMH-induced gene that is expressed in the Müllerian duct mesenchyme exclusively in male embryos. Osx-Cre transgenic mice were previously generated using a ∼204-kb BAC clone. Crosses of Osx-Cre mice to Cre-dependent lacZ reporter mice resulted in reporter expression in the developing skeleton and in the Müllerian duct mesenchyme of male but not female embryos. Osx-Cherry transgenic mice were previously generated using a 39-kb genomic region surrounding the Osx locus. Osx-Cherry embryos expressed red fluorescence in the developing skeleton and Müllerian duct mesenchyme of male but not female embryos. In addition, female Osx-Cherry embryos ectopically expressing human AMH from an Mt1-AMH transgene activated red fluorescence in the Müllerian duct mesenchyme. These results suggest that the 39-kb region used to generate Osx-Cherry contains male-specific Müllerian duct mesenchyme regulatory sequences that are responsive to AMH signaling. These BAC transgenic mouse approaches identify two distinct regions that direct Müllerian duct mesenchyme expression and contribute fundamental knowledge to define a gene regulatory network for sex differentiation.

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.

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.

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.

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.

Anti-Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extragonadal effects of AMH on the hypothalamic-pituitary-gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-dependent LH pulsatility and secretion, supporting a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of polycystic ovary syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings raise the intriguing hypothesis that AMH-dependent regulation of GnRH release could be involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

Plasma anti-Mullerian 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 Mullerian-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 (0), 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 Mullerian-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. (C) 2015 Elsevier Inc. All rights reserved.

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.

Anti-Müllerian hormone (AMH) is a member of the TGF-β superfamily secreted by the gonads of both sexes. This hormone is primarily known for its role in the regression of the Müllerian ducts in male fetuses. In females, AMH is expressed in granulosa cells of developing follicles. Like other members of the TGF-β 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 Müllerian 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-β 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.

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.

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. © 2008 Wiley‐Liss, Inc.