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Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.

International audience; In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.

Experimental data obtained in previous works have led to postulate that enhancers increase the frequency of action of a linked promoter in a given cell and may have some insulating effects. The multimerized rabbit alpha s1-casein gene enhancer, the 6i multimer, was added upstream of the rabbit whey acidic protein gene (WAP) promoter (-6,300; +28 bp) fused to the firefly luciferase (luc) gene (6i WAP-luc construct). The 6i multimer increased reporter gene expression in mouse mammary HC11 cells. In transgenic mice, a very weak but significant increase was also observed. More noticeable, no silent lines were found when the 6i multimer was associated to the WAP-luc construct. This reflects the fact that the 6i multimer tends to prevent the silencing of the WAP-luc construct. After addition of the 5'HS4 insulator region from the chicken beta-globin locus upstream of the 6i multimer, similar luciferase levels were measured in 6i WAP-luc and 5'HS4 WAP-luc transgenic mice. Our present data and previous ones, which show that the 6i multimer has no insulating activity on a TK gene promoter construct indicate that the insulating activity of the 6i multimer is construct-dependent and not amplified by the 5'HS4 insulator.

In vitro maturation of human oocytes at the germinal vesicle (GV) stage could offer an alternative in several cases of female infertility. It however rests on a better knowledge of the quality of human oocyte. Using fluorescence imaging of DNA and of the transcription sites, combined with electron microscopy, we show that human oocytes follow size-dependent changes in chromatin configuration, transcription sites distribution and nuclear ultrastructure that follow those observed in mouse GV oocytes. We thus analyzed in mouse GV oocytes the phosphorylation dependence of the transcriptional activity. We show by Western blot that, while active GV oocytes have approximately the same proportion of hypo- and hyperphosphorylated forms of the RNA polymerase II (RNAP II), the hyperphosphorylated form is almost absent from inactive oocytes. We also show that (1) RNAP II-dependent transcription is much less sensitive to various kinase inhibitors in mouse oocytes than in somatic cells or mouse one-cell embryos, although the phosphorylation equilibrium of RNAP II was largely shifted towards the hypo-phosphorylated form upon treatment with these inhibitors (2) RNAP I is completely insensitive to kinase inhibitors in GV oocytes.

Article online in advance of print; International audience; The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 µm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 µm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue. Mol. Reprod. Dev. 74: 989–996, 2007. © 2007 Wiley-Liss, Inc.

Gastrulation and trophectoderm elongation of the porcine conceptus coincide with peak conceptus estrogen secretion from gestational day 11 to day 12. The current study aim was to identify genes required for elongation by defining the transcriptome profile of this dynamic tubular stage. The gastrulation and proliferative status of ovoid, tubular, and filamen- tous conceptuses were also examined. Polarization of the embryonic disc and growth throughout the con- ceptus were evident. An unamplified and two distinct amplified serial analysis of gene expression (SAGE) libraries were generated from tubular conceptus mRNA. Comparing the three libraries at 12,000 tags/ library indicated small-amplified RNA-SAGE was a reliable amplification procedure. The unamplified library was increased to 42,415 tags and statistical analyses of tag frequencies with previously generated ovoid and filamentous libraries revealed the differential expression (P