In Vitro Fertilization and Embryo Culture Strongly Impact the Placental Transcriptome in the Mouse Model

BACKGROUND: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cell

H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions

Immature Cryopreserved Ovary Restores Puberty and Fertility in Mice without Alteration of Epigenetic Marks

BACKGROUND: Progress in oncology could improve survival rate in children, but would probably lead to impaired fertility and puberty. In pre-pubertal girls, the only therapeutic option is the cryopreservation of one ovary. Three births have been reported after reimplantation of cryopreserved mature ovary. Conversely, reimplantation of ovary preserved before puberty (defined as immature ovary) has never been performed in humans. METHODOLOGY/PRINCIPAL FINDINGS: In order to analyze ovarian function, we performed transplantation using fresh or cryopreserved immature grafts in pre-pubertal or adult mice. Puberty as well as cyclic hormonal activity was restored. All follicle populations were present although a significant reduction in follicle density was observed with or without cryopreservation. Although fertility was restored, the graft is of limited life span. Because ex vivo ovary manipulation and cryopreservation procedure, the status of genomic imprinting was investigated. Methylation status of the H19 and Lit1 Imprinting Control Regions in kidney, muscle and tongue of offsprings from grafted mice does not show significant alteration when compared to those of unoperated mice. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that immature ovarian grafting can restore spontaneous puberty and fertility. However, these data suggest that follicle depletion leads to premature ovarian failure. This study addresses the very important epigenetics issue, and provides valuable information to the study of ovarian transplantation suggesting that these procedures do not perturb normal epigenetics marks. These results are highly relevant to the reimplantation question of immature cortex in women

L'empreinte parentale

National audienc

Épigénétique et développement : l’empreinte parentale

Dans la nature, certaines espèces, animales comme végétales, se reproduisent par parthénogenèse,c’est-à-dire uniquement à partir du gamète femelle, sans participation du gamète mâle. Chez les mammifères, la parthénogenèse naturelle n’a jamais été observée, ce qui suggère que le développement d’un embryon de mammifèrerequiert la présence des deux génomes maternel et paternel. Cela est dû à un phénomène,découvert au début des années 1980, appelé «empreinte génomique parentale»: il apparaît que, chez tous les mammifères, les génomes mâle et femelle qui se rencontrent dans l’oeuf fécondé sont marqués d’un sceau différent,nommé empreinte. Par la suite, l’identification de gènes spécifiques soumis à empreinte parentalea permis de montrer que cette empreinte conduit à une expression monoallélique, dépendantede l’origine parentale. Les caractéristiques moléculaires de ce phénomène de marquage épigénétiqueont maintenant été décrites et permettentd’expliquer certaines maladies humaines liées à des gènes soumis à empreinte

The H19 locus: Role of an imprinted noncoding RNA in growth and development

International audienceThe H19 gene produces a non-coding RNA, which is abundantly expressed during embryonic development and down-regulated after birth. Although this gene was discovered over 20 years ago, its function has remained unclear. Only recently a role was identified for the non-coding RNA and/or its microRNA partner, first as a tumour suppressor gene in mice, then as a trans-regulator of a group of co-expressed genes belonging to the imprinted gene network that is likely to control foetal and early postnatal growth in mice. The mechanisms underlying this transcriptional or post-transcriptional regulation remain to be discovered, perhaps by identifying the protein partners of the full-length H19 RNA or the targets of the microRNA. This first in vivo evidence of a functional role for the H19 locus provides new insights into how genomic imprinting helps to control embryonic growth

Le locus IGF2/H19 (un modèle d'étude épigénétique du cancer colorectal)

Les modifications épigénétiques, telle que la méthylation de l'ADN sur les dinucléotides CpG, occupent une place importante en cancérogenèse. En particulier, les gènes IGF2 et H19 soumis à l'empreinte génomique parentale sont exposés à ces altérations de la méthylation et sont donc impliqués dans de nombreux cancers, incluant le cancer colorectal. Une étude du statut de méthylation des régions différentiellement méthylées (DMR) au niveau du locus IGF2/H19, nous a permis de mettre en évidence des dérégulations spécifiques des profils de méthylation des cancers colorectaux d'origine sporadique et héréditaire à la fois dans les tumeurs et dans les lymphocytes. Parallèlement, à l'aide d'un modèle murin du cancer colorectal, nous avons étudié le rôle du gène H19 dans la tumorigenèse colique. Cette étude confirme que le gène H19 agit en tant que gène suppresseur de tumeur chez la souris.Epigenetic modifications, such as DNA methylation on CpG sites, play an important role in carcinogenesis. In particular, IGF2 and H19 imprinted genes are exposed to these alterations of the methylation and are thus implied in numerous cancers, including colorectal cancer. By studying the methylation status of the differentially methylated regions (DMR) localized in the IGF2/H19 locus, we identified specific deregulations of the methylation patterns of colorectal cancers from sporadic and hereditary origin, both in tumors and lymphocytes. In parallel, using a colorectal cancer murine model, we studied the role of H19 gene in the colonic tumorigenesis. This study confirms that H19 gene acts as a tumor suppressor gene in mouse.PARIS5-BU Méd.Cochin (751142101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

H19 controls reactivation of the imprinted gene network during muscle regeneration

International audienceThe H19 locus controls fetal growth by regulating expression of several genes from the imprinted gene network (IGN). H19 is fully repressed after birth, except in skeletal muscle. Using loss-of-function H19(Delta 3) mice, we investigated the function of H19 in adult muscle. Mutant muscles display hypertrophy and hyperplasia, with increased Igf2 and decreased myostatin (Mstn) expression. Many imprinted genes are expressed in muscle stem cells or satellite cells. Unexpectedly, the number of satellite cells was reduced by 50% in H19(Delta 3) muscle fibers. This reduction occurred after postnatal day 21, suggesting a link with their entry into quiescence. We investigated the biological function of these mutant satellite cells in vivo using a regeneration assay induced by multiple injections of cardiotoxin. Surprisingly, despite their reduced number, the self-renewal capacity of these cells is fully retained in the absence of H19. In addition, we observed a better regeneration potential of the mutant muscles, with enhanced expression of several IGN genes and genes from the IGF pathway