Uterine defects and estradiol-dependent development of oviductal diverticula in mice lacking the SMAD4 C-terminal Mad homology 2 domain.

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.

Inactivation of Exosc10 in the oocyte impairs oocyte development and maturation, leading to a depletion of the ovarian reserve in mice.

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.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition.

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.

EXOSC10/Rrp6 is post-translationally regulated in male germ cells and controls the onset of spermatogenesis.

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.

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-ß) 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 ß-catenin conditional mutant mouse model, ß-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that ß-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/ß-catenin signalling leading to the partial persistence of Müllerian duct.

Combining RNA and protein profiling data with network interactions identifies genes associated with spermatogenesis in mouse and human.

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.

COUP-TFII mediates progesterone regulation of uterine implantation by controlling ER activity.

Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone-Indian hedgehog-Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.

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.

The regulation of COUP-TFII gene expression by Ets-1 is enhanced by the steroid receptor co-activators.

Recent phenotypic analysis of orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) [NR2F2] knockout mice shows that COUP-TFII is involved in the angiogenic process in the developing embryos. Since Ets-1 expression is also correlated with angiogenesis, and both Ets-1 and COUP-TFII mRNA are present in mesenchymal cells, we have sought to determine whether Ets-1 is a potential regulator of COUP-TFII gene expression. For this purpose, we performed transient transfection experiments using a luciferase reporter construct containing the mouse COUP-TFII promoter. We found that the COUP-TFII promoter activity is indeed regulated by Ets-1. We have identified two identical inverted potential ETS-binding sites located 47 nucleotides downstream of the start site. Mutation of both sites reduced the ability of Ets-1 to enhance the COUP-TFII promoter activity. Furthermore, other members of the ETS family such as Ets-2 or ETV1 are also potent regulators of the COUP-TFII promoter. Finally, the induction of the COUP-TFII gene is strongly enhanced by the expression of steroid receptor co-activator factors through a direct interaction with Ets-1. These results indicate that COUP-TFII is a potential downstream target of Ets-1 and it may partially mediate the Ets-1 function in angiogenesis.

Induction of chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI ) gene expression is mediated by ETS factor binding sites.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI, or NR2F1) is an orphan nuclear receptor that plays a major role in the development of the nervous system. We show here that three ETS response elements in the COUP-TFI promoter mediate its transcription. A reporter gene containing these ETS binding sites is activated by Ets-1, while the same reporter with point mutations on all three ETS response elements is not. We also show that Ets-1 binds to these response elements and that other ETS factors also transactivate the COUP-TFI promoter. In addition, COUP-TFI is coexpressed with some ETS factors in the mouse embryo. These results indicate that members of the ETS family can activate COUP-TFI gene expression.