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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.

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

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.

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.

This study was conducted to determine if the cadmium-mediated inhibition of vitellogenesis observed in fish collected from contaminated areas or undergoing experimental exposure to cadmium correlated with modification in the transcriptional activity of the estrogen receptor. A recombinant yeast system expressing rainbow trout (Oncorhynchus mykiss) estradiol receptor or human estradiol receptor was used to evaluate the direct effect of cadmium exposure on estradiol receptor transcriptional activity. In recombinant yeast, cadmium reduced the estradiol-stimulated transcription of an estrogen-responsive reporter gene. In vitro-binding assays indicated that cadmium did not affect ligand binding to the receptor. Yeast one- and two-hybrid assays showed that estradiol-induced conformational changes and receptor dimerization were not affected by cadmium; conversely, DNA binding of the estradiol receptor to its cognate element was dramatically reduced in gel retardation assay. This study provides mechanistic data supporting the idea that cadmium is an important endocrine disrupter through a direct effect on estradiol receptor transcriptional activity and may affect a number of estrogen signaling pathways.

De nombreuses publications font l'etat de l'action negative du cadmium (Cd ++ ) sur la vitellogenese chez les poissons. Le mecanisme d'action de ce metal n'etant pas clairement etabli, une etude a ete entreprise afin de determiner si l'effet du cadmium passait par une action sur l'activite biologique du recepteur des oestrogenes de la truite arc-en-ciel (rtER). Cette etude a ete realisee dans un systeme de levures recombinantes, exprimant de maniere stable rtER, et dans des hepatocytes de truite en culture primaire. Dans la levure, rtER active par l'oestradiol (E2) controle specifiquement l'expression d'un gene rapporteur codant pour l'enzyme β-Galactosidase. Une concentration en Cd ++ de 10 μM diminue de 45 % l'expression stimulee par l'oestradiol du gene rapporteur dans les levures recombinantes. De meme, dans les hepatocytes, l'expression du gene de la vitellogenine (VTG), gene sous le controle positif de E2, est diminuee de 65 %. Le cadmium n'affecte pas la liaison de E2 a son recepteur et seule l'activite hormono-dependante est affectee par ce metal. Les resultats de cette etude montrent que l'effet negatif du cadmium sur l'action de l'oestradiol passe vraisemblablement par une action directe de ce metal sur la fonction de transactivation hormono-dependante de rtER.

Rainbow trout estrogen receptor (rtER) concentration was highly induced in the liver after in vivo estradiol (E2) treatment or in vitro, in hepatocyte aggregate culture. Determination of transcription rate and mRNA half-life demonstrated that E2-induction of hepatic rtER level is caused essentially by an increase in the transcriptional and post-transcriptional activity of rtER gene. However, the expression of rtER gene in the liver seems to be down-regulated by glucocorticoids. We have used transient transfection assays with reporter plasmids linked to 5′ flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized a functional estrogen-responsive-element (ERE), near the transcription start site, with one mutation on the first base compared to the consensus sequence. This element and 200 bp fragment of the rtER promoter encompassing the ERE appear to be the major cis-acting element involved in the regulation of the gene. Data obtained from transfection experiments and footprinting analysis, suggested that the receptor is one of the major trans-factors implicated in the regulation of its own gene. However, interaction of ER with other transcription factors is required for maximal E2-stimulation.

The cDNA of rainbow trout estrogen receptor (rtER), highly and stably expressed in yeast, Saccharomyces cerevisiae, was used to analyse the biological activity of the receptor. The rtER mRNA encoded a 65-kDa protein which was immunorevealed by a specific antibody and migrated with the authentic rtER major protein form detected in trout liver. Yeast rtER bound estradiol with high affinity and the dissociation constant (Kd = 1.35 nM) was very similar to the value measured from trout liver extracts but 3-5-fold higher than the Kd found for human estrogen receptor (hER). This indicates therefore that the rtER has a lower estradiol affinity compared to the human receptor. While the hER Kd remained unchanged at both 4 degrees C or 22 degrees C, it was slightly modified at 30 degrees C. The Kd measured for rtER at 22 degrees C and 30 degrees C were about 2-fold, and 12-fold higher, respectively, than the Kd obtained at 4 degrees C suggesting an alteration of the rtER affinity for its ligand at elevated temperature. To examine the estrogen-receptor-mediated activation of transcription in yeast, reporter plasmids integrated or not in the yeast genome were used. The reporter genes consist of one, two, or three copies of estrogen-responsive elements (ERE) upstream of the yeast proximal CYC1 or URA3 promoters fused to the lacZ gene of Escherichia coli coding for beta-galactosidase. The induction of beta-galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens. The activation of transcription mediated by rtER responded in an estradiol-dose-dependent manner as in animal cells. However, compared to hER, the estradiol concentration necessary to achieve maximal activation was 10-fold higher. This is probably a consequence of the lower estradiol-affinity for rtER compared to hER. The levels of induction of the reporter genes containing two or three ERE were strongly enhanced compared to the one ERE construct. This is in agreement with the synergistic effect previously described for multiple ERE. The magnitudes of transcriptional induction mediated by rtER and hER were similar when the reporter gene containing three ERE was used but changed when the one ERE construct was used. In this case transcriptional activation indicated by rtER was 10-20 fold lower. This suggests that rtER requires protein/protein interaction for its stabilization on DNA. Antiestrogens were able to bind rtER and promote gene transcription. However, to produce effects comparable to those obtained with estrogens, much higher concentrations were required. This may imply nonetheless that antihormones were capable of provoking efficient interactions of rtER with the transcriptional machinery.

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.

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., Author Summary Pregnancy is established and maintained through a series of precisely choreographed cellular and molecular events that are controlled by two sex hormones, estrogen and progesterone. Both hormones exert their actions through their distinct nuclear receptors. During the peri-implantation period, estrogen activity is attenuated by progesterone to facilitate epithelial remodeling and embryo attachment, but the detailed molecular mechanism of how this process is achieved remains largely undefined. 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 controlled by progesterone–Indian hedgehog–Patched signaling from the epithelium to the stroma. To assess the uterine function of COUP-TFII, uterine-specific COUP-TFII knockout mice were generated. These mutant mice are infertile due to failure of implantation. We identified a novel genetic pathway in which the epithelial Ihh regulates the stroma COUP-TFII to control BMP2 and regulates decidualization. Interestingly, enhanced epithelial estrogen activity, which impedes the maturation of receptive uterus, was clearly noted in the absence of COUP-TFII. This finding reveals that COUP-TFII plays a critical role in maintaining the balance between estrogen and progesterone activities to establish proper implantation. This finding also provides new insights into women's health care associated with uncontrolled estrogen activity, such as breast cancer and endometriosis.