The 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.

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss-and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.

The H19 locus belongs to a cluster of imprinted genes that is linked to the human Beckwith-Wiedemann syndrome. The expression of H19 and its closely associated IGF2 gene is frequently deregulated in some human tumors, such as Wilms' tumors. In these cases, biallelic IGF2 expression and lack of expression of H19 are associated with hypermethylation of the imprinting center of this locus. These observations and others have suggested a potential tumor suppressor effect of the H19 locus. Some studies have also suggested that H19 is an oncogene, based on tissue culture systems. We show, using in vivo murine models of tumorigenesis, that the H19 locus controls the size of experimental teratocarcinomas, the number of polyps in the Apc murine model of colorectal cancer and the timing of appearance of SV40-induced hepatocarcinomas. The H19 locus thus clearly displays a tumor suppressor effect in mice.genomic imprinting ͉ Igf2 ͉ murine models T he H19-Igf2 locus is subject to genomic imprinting and has often been used as a paradigm for the study of this particular epigenetic regulation. The H19 locus produces a 2.5-kb noncoding, spliced, and polyadenylated RNA of yet-unknown function (1, 2). The Igf2 gene encodes a fetal growth factor, insulin-like growth factor 2. These two genes are located 90 kb apart and are oppositely imprinted: H19 is maternally expressed and Igf2 paternally expressed (1, 3). They belong to a large imprinted domain localized on chromosome 7 in mice and chromosome 11p15.5 in humans. The imprinting of Igf2 and H19 is controlled by a region located 4 kb upstream from the H19 transcription unit, defined as the H19 differentially methylated region (DMR) or imprinting control region (ICR) (4).The 11p15.5-imprinted domain is linked to the BeckwithWiedemann syndrome (BWS), which is characterized by overgrowth phenotypes of affected children as well as a predisposition to develop embryonal tumors such as Wilms' tumor or rhabdomyosarcomas (5). Among the molecular alterations involved in BWS, certain cases (20%) show paternal uniparental disomy (UPD); other cases (5-10%) have hypermethylation of the H19 DMR; and both lead to lack of expression of H19 as well as activation of IGF2. These patients have higher risk of developing tumors than patients with other molecular defects (6). Genetic and epigenetic alterations at 11p15.5 similar to those found in the BWS have also been demonstrated in nonsyndromic Wilms' tumors. A great number of these cases have either loss of the maternal allele (LOH) or LOI (7,8). It has thus been suggested that the H19 gene could have a possible tumor suppressor function (9). The first direct evidence for this tumor suppressor function was provided by in vitro experiments in which transfection of H19 cDNA into G401-transformed kidney cells resulted in loss of tumorigenicity of these cells (10). Subsequent experiments performed with in vitro culture systems suggested that H19 played a role as an oncogene rather than a tumor suppressor gene (11,12). This controversy has not yet been reso...

DNA methylation marks, a key modification of imprinting, are erased in primordial germ cells and sex specifically re-established during gametogenesis. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. We analysed by pyrosequencing the DNA methylation status of 47 CpGs located in differentially methylated regions (DMRs), the DMR0 and DMR2 of the IGF2 gene and in the 3rd and 6th CTCF-binding sites of the H19 DMR in human sperm from men with normal semen and patients with teratozoospermia (T) and/or oligo-astheno-teratozoospermia (OAT). All normal semen samples presented the expected high global methylation level for all CpGs analysed. In the teratozoospermia group, 11 of 19 patients presented a loss of methylation at variable CpG positions either in the IGF2 DMR2 or in both the IGF2 DMR2 and the 6th CTCF of the H19 DMR. In the OAT group, 16 of 22 patients presented a severe loss of methylation of the 6th CTCF, closely correlated with sperm concentration. The methylation state of DMR0 and of the 3rd CTCF was never affected by the pathological status of sperm samples. This study demonstrates that epigenetic perturbations of the 6th CTCF site of the H19 DMR might be a relevant biomarker for quantitative defects of spermatogenesis in humans. Moreover, we defined a methylation threshold sustaining the classification of patients in two groups, unmethylated and methylated. Using this new classification of patients, the observed intrinsic imprinting defects of spermatozoa appear not to impair significantly the outcome of assisted reproductive technologies.

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Background: In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos.

-Secretions collected from the mammary gland of different species contain heterogeneous populations of cells including lymphocytes, neutrophils, macrophages and epithelial cells in different species. Several factors influence the somatic cell count in milk and the distribution of cell types, such as species, infection status, physiological status and management practices. The epithelial cells are shed into milk during the lactation process. Most of them are viable and exhibit the characteristics of fully differentiated alveolar cells. Primary cultures of epithelial cells from colostrum and milk of humans, baboons, cows and goats together with established cell lines from human and goat milk, provide a good model for the study of lactogenesis, immunity transmission, cancer research and infection by viruses. The RNA extracted from milk cells have been shown to be representative of gene expression in the mammary gland and thus provide a source of material for molecular studies of gene expression and environmental interactions. milk / somatic cell count / epithelial cellsRésumé -Utilisations potentielles des cellules épithéliales du lait. Les sécrétions de la glande mammaire de différentes espèces, contiennent une population hétérogène de cellules incluant des cellules du système immunitaire et des cellules épithéliales. Divers facteurs influencent le nombre de cellules somatiques dans le lait et la distribution de chaque type cellulaire, en particulier l'espèce, le stade physiologique, l'état sanitaire et les conduites d'élevage. Les cellules épithéliales se détachent de l'épi-thélium mammaire au cours du processus de sécrétion du lait. Cependant, une grande majorité d'entre elles sont viables et présentent des caractéristiques de cellules alvéolaires totalement différenciées. En culture primaire ou en lignées cellulaires, elles constituent un modèle d'étude de l'action hormonale, de marqueurs de la cancérogenèse, de la transmission de l'immunité et d'infections virales. Les ARN totaux extraits des cellules du lait constituent une source de matériel représentatif de l'expression des gènes dans la glande mammaire et devraient permettre des études globales d'expression des gènes en interaction avec l'environnement de l'animal. lait / numération cellulaire / cellule épithéliale Reprod. Nutr. Dev. 42 (2002) 133-147 133

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