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It has been clearly demonstrated that the maternal nutritional status during pregnancy and lactation has long-term effects on offspring health. In mammals, milk represents the first maternal support provided to the newborns so that its composition may play a major role in long-term programming. We therefore assessed the effects of maternal high-fat/high-sugar obesogenic (OD) or control (CD) diets on offspring growth and adiposity in the rabbit. Between 7 and 20 wk of age, the BW gain of OD milk-fed rabbits was higher than that of CD milk-fed rabbits ( < 0.05). Body fat mass measurements at 21 wk of age revealed a significant increase in body adiposity as a function of milk ingested during the neonatal period, in both female and male offspring ( < 0.05). A marked weight gain difference was observed according to the milk in both female and male offspring. Moreover, we investigated the composition in major proteins and leptin levels in milk from OD or CD diet-fed dams. Liquid chromatography-mass spectrometry analysis of individual CD skimmed milk samples enabled identification and quantification of the rabbit main milk proteins and of their main phosphorylated isoforms at 2 different stages of lactation (3 and 10 d). Here we show that the OD diet induced a reduction in the whey acidic protein content concomitantly with both an increase in serum albumin and lactoferrin contents and in the phosphorylated isoforms of the main milk proteins. Furthermore, a sharp rise in leptin levels was observed in the milk of OD diet-fed dams on Day 10 of lactation when compared with CD diet animals ( < 0.05). Taken together, these findings provide evidence that lactation is a critical window of development during which exposure to a deleterious diet is highly detrimental to long-term outcomes. Moreover, these insights suggest that it may be possible to prevent at least some of the adverse effects of inadequate maternal nutrition on the long-term metabolic outcomes of the offspring through nutritional interventions applied during the lactation period.

Alterations to the metabolic endocrine environment during early life are crucial to mammary gland development. Among these environmental parameters, the initial nutritional event after birth is the consumption of milk, which represents the first maternal support provided to mammalian newborns. Milk is a complex fluid that exerts effects far beyond its immediate nutritional value. The present study, therefore, aimed to determine the effect of the nutritional changes during the neonatal and prepubertal periods on the adult mammary phenotype. Newborn rabbits were suckled by dams fed a high-fat/high-sugar obesogenic (OD) or a control (CON) diet and then subsequently fed either the OD or CON diets from the onset of puberty and throughout early pregnancy. Mammary glands were collected during early pregnancy (Day 8 of pregnancy). Rabbits fed with OD milk and then subjected to an OD diet displayed an abnormal development of the mammary gland: the mammary ducts were markedly enlarged ( < 0.05) and filled with abundant secretory products. Moreover, the alveolar secretory structures were disorganized, with an abnormal aspect characterized by large lumina. Mammary epithelial cells contained numerous large lipid droplets and exhibited fingering of the apical membrane and abnormally enlarged intercellular spaces filled with casein micelles. Leptin has been shown to be involved in modulating several developmental processes. We therefore analyzed its expression in the mammary gland. Mammary leptin mRNA was strongly expressed in rabbits fed with OD milk and subjected to an OD diet by comparison with the CON rabbits. Leptin transcripts and protein were localized in the epithelial cells, indicating that the increase in leptin synthesis occurs in this compartment. Taken together, these findings suggest that early-life nutritional history, in particular through the milking period, can determine subsequent mammary gland development. Moreover, they highlight the potentially important regulatory role that leptin may play during critical early-life nutritional windows with respect to long-term growth and mammary function.

The rabbit kappa-casein encoding gene has previously been shown to possess two alleles. The two alleles do not differ in their coding region and in the accumulation levels of mRNA. However they differ greatly with respect to their intronic regions. The rearranged regions in the first and fourth introns were found to be inverse and complementary LINE sequences. The A allele was found to be more frequent in different European breeds. Correlation of the kappa-casein genotype with the breeding capacity in a New Zealand White rabbit stock has been examined.

Once-daily milking is known to modify cell number and activity in the bovine mammary gland. However, in goats, the effect of once-daily milking on mammary cell death is controversial. To assess the effect of once-daily milking on mammary transcriptome and cell turnover and the effect of the duration of once-daily milking, 10 goats producing 2.5 kg of milk per day at 100 DIM were divided into 2 groups. All goats were twice daily milked during a pre-experimental period of 2 wk. Then, the goats were once daily milked for the 3 following weeks. Mammary biopsy samples were collected at d −1 and 7 after the start of once-daily milking for one group of 5 goats and at d −1 and 21 for a second group of 5 goats. Cell apoptosis and proliferation rates were analyzed in mammary tissue by immunohistological analyses after TUNEL and PCNA staining, respectively. Ribonucleic acid was extracted from mammary tissues. A transcriptomic analysis using the Agilent Bovine 4x44k microarrays has been performed to compare the effect of once-daily milking on mammary transcript profiles. Data were normalized and statistical significant raw P-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. A similar reduction in milk yield (−19%) was observed in both groups of goats during once-daily milking compared with twice-daily milking. Cell apoptosis was higher during once-daily milking than during twice-daily milking for both groups of goats (P < 0.05) whereas cell proliferation did not vary (P = 0.27). The transcriptomic analysis showed a differential gene expression of 4,039 transcripts, 2,238 and 1,801 transcripts up- and downregulated, respectively, by once-daily milking compared with twice-daily milking. More than 1,000 transcripts were commonly regulated between the 2 groups of goats. IPA analysis showed that these transcripts were part of networks associated with DNA replication, cellular growth and proliferation, and cell-to-cell signaling and interaction in both groups of goats. RT-qPCR analysis of 11 genes confirmed the differential gene expression with a downregulation of genes involved in milk synthesis and an upregulation of genes involved in cellular cycle and apoptosis. These results showed that once-daily milking induce cell turnover in goat mammary tissue, with a small impact of time.

Alterations to the metabolic environment during puberty can impact future lactation efficiency and mammary tumorigenesis. During this study, we used a model of rabbits receiving an obesogenic diet (OD), starting before puberty and extending until mid-pregnancy. Three months later, the body weight of OD animals was significantly higher than that of controls and their mammary glands displayed a precocious and abnormal development at mid-pregnancy. OD mammary ducts were filled with dense products, while alveolar structures invaded most of the fat pad. The proportion of secretory epithelium was significantly higher in OD mammary tissue, which contained an abundant accumulation of milk proteins and lipids. In conclusion, an obesogenic diet started before puberty induced an accelerated development of the rabbit mammary gland, leading to an accumulation of secretory products at mid-pregnancy. These results support the critical influence of nutrition on mammary growth and differentiation, which may be deleterious to mammary development and subsequent lactation. Developmental Dynamics 240:347–356, 2011. © 2011 Wiley-Liss, Inc.

International audience; Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein. Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of β-casein is post- translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway.

International audience; Compartmentalization is one of the fundamental principles which underly nuclear function. Numerous studies describe complex and sometimes conflicting relationships between nuclear gene positioning and transcription regulation. Therefore the question is whether topological landmarks and/or organization principles exist to describe the nuclear architecture and, if existing, whether these principles are identical in the animal and plant kingdoms. In the frame of an agroBI-INRA program on nuclear architecture, we set up a multidisciplinary approach combining biological studies, spatial statistics and 3D modeling to investigate spatial organization of a nuclear compartment in both plant and animal cells in their physiological contexts. In this article, we review the questions addressed in this program and the methodology of our work.; Modéliser l'architecture tri-dimensionnelle du noyau dans des cellules animales et végétales. L'organisation en comparti-ments est un des principes fondamentaux qui gouverne le fonctionnement du noyau. De nombreuses études montrent des relations complexes et parfois contradictoires entre la position d'un gène dans le noyau et son état transcriptionnel. Deux questions se posent : existe-t-il des principes généraux gouvernant l'organisation nucléaire, et, si oui, ces principes sont-ils analogues dans les règnes animal et végétal ? Dans le cadre d'un programme agroBI-INRA sur l'architecture nucléaire, nous avons développé une ap-proche multidisciplinaire combinant expériences biologiques, statistiques spatiales et modélisation en 3 dimensions pour analyser l'organisation spatiale d'un compartiment nucléaire dans des cellules animales et végétales, dans leur environnement physiolo-gique. Dans cet article, nous faisons une revue autour des questions posées dans ce programme et présentons la méthodologie de ce travail

International audience; Whey acidic protein (WAP) and casein (CSN) genes are among the most highly expressed milk protein genes in the mammary gland of the lactating mouse. Their tissue-specific regulation depends on the activation and recruitment of transcription factors, and chromatin modifications in response to hormonal stimulation. We have investigated if another mechanism, such as specific positioning of the genes in the nucleus, could be involved in their functional regulation. Fluorescent in situ hybridization was used to study the nuclear localization of WAP and CSN genes in mouse mammary epithelial cells (HC11) cultured in the absence and presence of lactogenic hormones. Automatic 3D image processing and analysis tools were developed to score gene positions. In the absence of lactogenic hormones, both genes are distributed non-uniformly within the nucleus: the CSN locus was located close to the nuclear periphery and the WAP gene tended to be central. Stimulation by lactogenic hormones induced a statistically significant change to their distance from the periphery, which has been described as a repressive compartment. The detection of genes in combination with the corresponding chromosome-specific probe revealed that the CSN locus is relocated outside its chromosome territory following hormonal stimulation, whereas the WAP gene, which is already sited more frequently outside its chromosome territory in the absence of hormones, is not affected. We conclude that milk protein genes are subject to nuclear repositioning when activated, in agreement with a role for nuclear architecture in gene regulation, but that they behave differently as a function of their chromosomal context.

International audience; Lactation performance is dependent on both the genetic characteristics and the environmental conditions surrounding lactating cows. However, individual variations can still be observed within a given breed under similar environmental conditions. The role of the environment between birth and lactation could be better appreciated in cloned cows, which are presumed to be genetically identical, but differences in lactation performance between cloned and noncloned cows first need to be clearly evaluated. Conflicting results have been described in the literature, so our aim was to clarify this situation. Nine cloned Prim' Holstein cows were produced by the transfer of nuclei from a single fibroblast cell line after cell fusion with enucleated oocytes. The cloned cows and 9 noncloned counterparts were raised under similar conditions. Milk production and composition were recorded monthly from calving until 200 d in milk. At 67 d in milk, biopsies were sampled from the rear quarter of the udder, their mammary epithelial cell content was evaluated, and mammary cell renewal, RNA, and DNA were then analyzed in relevant samples. The results showed that milk production did not differ significantly between cloned and noncloned cows, but milk protein and fat contents were less variable in cloned cows. Furthermore, milk fat yield and contents were lower in cloned cows during early lactation. At around 67 DIM, milk fat and protein yields, as well as milk fat, protein, and lactose contents, were also lower in cloned cows. These lower yields could be linked to the higher apoptotic rate observed in cloned cows. Apoptosis is triggered by insulin-like factor growth binding protein 5 (IGFBP5) and plasminogen activator inhibitor (PAI), which both interact with CSN1S2. During our experiments, CSN1S2 transcript levels were lower in the mammary gland of cloned cows. The mammary cell apoptotic rate observed in cloned cows may have been related to the higher levels of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) transcripts, coding for products that maintain the epigenetic status of cells. We conclude, therefore, that milk production in cloned cows differs slightly from that of noncloned cows. These differences may be due, in part, to a higher incidence of subclinical mastitis. They were associated with differences in cell apoptosis and linked to variations in DNMT1 mRNA. However, milk protein and fat contents were more similar among cloned cows than among noncloned cows.

One of the major advances in the study of endocrine regulation of the mammary gland has been the development of techniques to obtain defined mutations in mice. The use of mice deficient in hormones, growth factors, receptors or transcription factors now allow to dissect hormonal pathways and to elucidate the specific role of the epithelium and the stroma. The study of temporal changes of global gene expression at each physiological stage is now possible by microarrays or proteomic analyses to profile gene or protein expressions, respectively. These approaches have been successful in demonstrating the huge number of genes that are differentially expressed in the murine and bovine mammary epithelial cells during pregnancy, lactation and involution. The next step to get the temporal and spatial assignement of gene function necessitate now the coordination of these technologies with accurate cellular biology approaches through in vivo and dynamic studies.

7 pages.-- PMID: 16180729 [PubMed]., For 10 years, the regulatory regions of the mouse and rabbit whey acidic protein gene have been used to express heterologous proteins in the milk of transgenic mice, as well as to produce pharmaceutical proteins, on a large scale, in the milk of transgenic livestock. To date, a broad range of expression levels have been detected, and elucidation of the structure-function relationship in these regulatory regions might help to achieve high levels of expression, reproducibly. An extended 5′ regulatory region (17·6 kb v. 6·3 kb) of the rabbit whey acidic promoter resulted in an increased frequency of rabbit whey acidic protein expression in transgenic mice. However, the expression levels were low compared with the high expression levels achieved in both transgenic mice and rabbits using the heterologous κ-casein in the 6·3 kb rabbit whey acidic protein 5′ regulatory region. These results underline the importance of the 3’ downstream regulatory regions, which still need to be better characterized in the whey acidic protein gene.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p

Séance: Régulation épigenetique: applications en élevage; Les phénotypes observés chez les animaux de rente sont déterminés en partie par le génome qui a fait l’objet d’une exploration produisant des quantités massives d’informations génomiques intégrées dans la prédiction de mérite génétique avec une grande exactitude. Cependant, un nouveau champ d’investigation a révélé l’importance de prendre en compte des mécanismes épigénétiques, qui peuvent refléter des effets environnementaux importants et améliorer notre compréhension de la construction des phénotypes. L’épigénétique se réfère aux changements héritables de l’activité génique en l’absence de toute modification de la séquence de l’ADN génomique. Des mécanismes moléculaires sous-jacents orchestrent la réorganisation de chromatine contrôlant ainsi la transcription des gènes. Ici, nous fournissons des exemples tirés de la littérature scientifique publiée soulignant que l’apposition des marques épigénétiques sur le génome peut être séquentielle, réversible et/ou héritable. Ces marques jouent un rôle majeur dans de nombreux processus biologiques tels que la différenciation gamétique, le développement de l’embryon ou encore la différenciation et le développement fonctionnel de la glande mammaire. Cette revue soulignera que le phénotype d’un individu est la résultante d’interactions complexes entre le génotype et l’environnement façonnant tout au long de la vie l’épigénome. Les marques épigénétiques constituent alors une véritable mémoire des événements de la vie incluant la vie in utero et assurent l’intégration multi-générationnelle et trans-générationnelle des effets de l’environnement. Nous proposons ainsi d’intégrer les informations concernant l’état de l’épigénome et de les considérer comme de nouvelles variables dans la sélection pour préserver la durabilité de l’élevage.

Nitric oxide (NO) is involved in the transmission of light information to suprachiasmatic nuclei (SCN). By immunocytochemistry, we showed that both neuronal and endothelial NO synthase isoforms (nNOS and eNOS) were present in the SCN of rats and hamsters. nNOS-immunoreactive neurons were located mainly around the SCN with only a few nNOS neurons within the nucleus. By double-label immunocytochemistry, we also found, within the population of SCN glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes, a subpopulation of eNOS-immunoreactive astrocytes. Using Western blot analysis, we detected in SCN protein extracts eNOS and nNOS proteins having the expected 140 and 150 kDa molecular weights, respectively. By in situ hybridization of a 2.4-kb murine eNOS probe, mRNA for eNOS was located in the SCN of rats and hamsters. The transcript was further identified by detection of a RT-PCR product of the predicted size, after amplification of total RNA with primers specific for eNOS. In the SCN and cerebellum, the size of the mRNA for nNOS, detected with a rat probe on Northern blot, was approximately 10.5 kb, corresponding to that previously published. In the same tissues, we found two transcripts, one weakly expressed at approximately 4.0 kb and another more strongly expressed at approximately 2.6 kb, both hybridizing with two non-overlapping murine and rat eNOS probes. These results suggested the existence in the SCN of alternate transcripts for eNOS. We propose that two pathways could link light stimuli and NO release in the SCN: one involving N-methyl-D-aspartate (NMDA) receptors and nNOS in neurons; the other linking alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and eNOS in astrocytes.

In a previous study we showed the existence of GH in the ovine placenta. We now supplement the information available on placental GH and describe the presence and distribution of GH receptor (GH-R) messenger RNA (mRNA) in uterine, fetal, and placental tissues during early pregnancy. GH mRNA was not detected in the placenta before day 27 (d27). Its expression peaked between d40 and d45 and fell after d55. GH mRNA was localized in the trophectoderm and syncytium. During the d35-d50 period, concentrations of GH in the maternal circulation were not increased. In umbilical blood, however, GH was detected from d35 and was presumed to be of placental origin, because GH mRNA was not detected in the fetal pituitary gland on d40. We report on GH-R mRNA expression in the placenta between d20-d120. The relative abundance of GH-R transcripts increased significantly between d25-d43. In the endometrium, GH-R mRNA was detected from d8-d120 of pregnancy and from d4-d16 of the cycle. GH-R mRNA was localized in the trophectoderm, fetal mesoderm, and maternal uterine stroma. In the fetal liver, GH-R mRNA was first detectable on d35. The results of this study indicate that between d35-d50 of pregnancy, the endometrium, placenta, and fetus are all potential targets for the placental GH. (Endocrinology 140: 5587‐ 5597, 1999)

The most frequent allele of the rabbit kappa-casein (kappa-Cas)-encoding gene (A allele) has previously been shown to possess two sequences similar to those found in the 5' end of long interspersed repeated elements (LINE). Part of an inverted rabbit LINE is present in the first intron and part of a direct rabbit LINE in the fourth intron. We describe herewith a less frequent allele (B allele) that lacks both 100bp in the first intron and 1550bp in the fourth intron. It was not possible to identify any allele exhibiting only one of the deletions in a population of 55 rabbits. The 100bp present in the first intron of the A allele, but absent from the B allele, are located at the 5' end of the inverse complementary LINE and include the poly (T) track of the LINE. The 1550bp present in the fourth intron of the A allele, but absent from the B allele, include the entire direct LINE sequence. Therefore, the B allele only possesses one partial LINE sequence that is located in the first intron and is truncated when compared to the copy found in the first intron of the A allele. The B allele might thus be more recent than the A allele. Differences between the sequences of transcripts corresponding to each allele are limited to two silent mutations and three modifications in the 3' UTR. In the mammary glands of lactating rabbits, which are homozygous for both alleles, kappa-Cas mRNA accumulate to similar levels and are translated into identical kappa-Cas that are secreted at similar concentrations into milk.

Introduction et but de l’etude Les donnees recentes indiquent que plus d’un adulte sur trois dans le monde, soit 1,46 milliard de personnes, est obese ou en surcharge ponderale. Cette obesite va avoir des consequences deletaires sur la sante des individus et en particulier sur l’allaitement des femmes ainsi que sur la composition de leur lait. A plus long terme, une alimentation maternelle inappropriee peut influer sur le developpement du fœtus et la sante de l’enfant, puis de l’adulte. Dans des modeles animaux, il a ete montre que l’engraissement excessif, en particulier pendant la puberte, peut induire des perturbations du developpement de la glande mammaire resultant a une baisse des capacites laitieres. L’objectif de notre etude est de determiner des fenetres critiques de sensibilite a l’alimentation en ciblant la periode neonatale via le lait, pouvant impacter la croissance et le developpement mammaire. Materiel et methodes Dans ces etudes, nous avons utilise des lapines nourries avec un regime obesogene (OB) enrichi en sucres (+300 %) et en graisses (+300 %). Dans une premiere cohorte, le regime a ete commence des la puberte. Les consequences d’une adiposite elevee sur la lactation ont ete etudiees sur une nouvelle cohorte de lapines suivant le meme protocole experimental. Afin de tester l’effet du lait sur la descendance, nous avons allaite les lapereaux des leur naissance avec un lait issu de meres ayant ete alimentees soit avec un regime OB, soit avec un regime temoin. A la puberte, ces lapereaux ont eux-memes ete soumis soit a un regime OB, soit au regime temoin. La croissance et l’adiposite de ces lapereaux ont ete suivies. Les glandes mammaires de ces animaux ont ete analysees sur le plan morphologique et fonctionnel. Resultats et Analyse statistique Trois mois apres le debut du regime, le poids des lapines ayant recu le regime OB etait significativement plus eleve. Cet ecart resultait d’une difference dans l’adiposite entre les deux groupes d’animaux. Les glandes mammaires des lapines OB presentaient un developpement modifie tant sur le plan morphologique que sur le plan fonctionnel : les canaux mammaires etaient remplis avec des produits denses. La proportion d’epithelium secretoire etait plus importante dans le tissu mammaire de ces animaux et l’analyse histologique de l’ epithelium mammaire a revele une abondante accumulation de proteines du lait et de lipides, rappelant un phenotype de fin de gestation. De plus, la composition du lait issu de ces lapines etait alteree. Lorsque ce lait a ete administre a des lapereaux, ceux-ci ont presente une predisposition a l’obesite caracterisee par une adiposite plus elevee. Lorsqu’il a ete combine a un regime obesogene, il a induit un developpement mammaire anormal des les stades precoces de la gestation : desorganisation des structures alveolaires, presence de canaux hyper-dilates contenant des proteines du lait et des globules gras, alteration de la polarite des cellules epitheliales. Conclusion Nos resultats demontrent qu’une alimentation enrichie en graisses et en sucres, commencee des la puberte tend a favoriser un developpement precoce et donc anormal de la glande mammaire. Ce type d’alimentation altere egalement la qualite du lait lors de la lactation. Le fait de consommer ce lait a la composition alteree aurait ainsi comme consequence d’induire une predisposition a une hyperadiposite et de potentialiser les effets nefastes d’un regime obesogene au niveau du developpement mammaire chez la descendance.

While microRNAs represent a well studied part of the non-coding genome, long non-coding transcripts are much more intricate, and are likely to contain as yet unidentified classes of molecules forming transcriptional regulatory networks. Long non-coding RNAs (ncRNAs) have long been considered as negative regulators, promoting chromatin silencing, but recently, several studies pointed out a role for a class of long ncRNAs in positive regulation of protein coding genes (Ørom et al., 2010, Lai et al., 2013) . In order to identify long ncRNAs that could be involved in testis or ovary differentiation, a high throughput RNA sequencing project was initiated in the bovine species. Strand-orientated libraries were prepared from transcripts of early testes and ovaries and next generation sequencing technologies (NGS) were used to identify all RNAs (coding and non-coding) expressed at this early stage in gonads (RNA-sequencing). As a first part, our attention has been focused on loci known to enclosed sex determining genes, to highlight putative regulatory function of long ncRNAs. Indeed, these NGS data may participate to the characterization of crucial regulatory regions. In addition, large scale characterization of all long non-coding RNAs differentially expressed in fetal ovaries or testes is in progress. Up to now, 5242 loci (coding and non-coding) have been showed to be differentially transcribed (pValue

Milking regulates both milk secretion and synthesis and its frequency in ruminants has been shown to have long lasting effects on milk production. The mechanisms involve a number of genes that are differentially expressed (1, 2). In this study, we hypothesized that a decrease in milking frequency induces methylation of CpG located within regulatory regions of genes expressed in the mammary gland. This hypothesis has been challenged on a candidate gene, the αS1-casein (CSN1S1 - a major milk protein) for which a distal regulatory region had been previously described (3, 4). We used an original animal model which allows studying epigenetic alterations within identical genetic and environment backgrounds. Milk production from the left and right udeer halves of eight heifers were studied over three periods of 7 days each. During the first and last periods, both udder halves were milked twice daily for 5 days, whereas during the second period, the right and left udder halves were differentially milked twice and once a day, respectively. Once daily milking (ODM) reduced the accumulation levels in the mammary gland of the main milk protein mRNAs (RNASeq), which represent up to 70% of mRNA expressed during lactation. It slightly increased the methylation level of the whole genome in the mammary gland (75.9%), as compared to that observed after twice daily milking (TDM); (74.6%, P=0.04); (LUMA). Simultaneously, it decreased the CSN1S1 mRNA level by 50%. The methylation level of four CpG sites located close to the distal regulatory region of this gene was evaluated in relation to gene expression. After ODM, the methylation level of the three more distal CpGs (relative to the transcription start point of the CSN1S1 gene) is higher as compared to that observed after TDM (P

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International audience; Leptin is known as a cytokine mostly produced by fat cells and implicated in regulation of energy metabolism and food intake but has also been shown to be involved in many physiological mechanisms such as tissue metabolism and cell differentiation and proliferation. In particular, leptin influences the development of mammary gland. Although leptin expression in mammary gland has been studied in several species, no data are available in the rabbit. Leptin transcripts in this species have been described as being encoded by only two exons rather than three as in other species. Our focus was to clone and sequence the rabbit leptin cDNA and to prepare the recombinant biologically active protein for validation of the proper sequence and then to describe leptin expression in rabbit mammary gland during different stages of pregnancy and lactation. The leptin sequence obtained was compared with those of other species, and genome alignment demonstrated that the rabbit leptin gene is also encoded by three exons. Additionally, we analyzed the expression of leptin during pregnancy and lactation. Leptin mRNA was weakly expressed throughout pregnancy, whereas mRNA levels were higher during lactation, with a significant increase between days 3 and 16. Leptin transcripts and protein were localized in luminal epithelial cells, thus indicating that leptin synthesis occurs in this compartment. Therefore, mammary synthesized leptin may constitute a major regulator of mammary gland development by acting locally as an autocrine and/or paracrine factor. Furthermore, our results support the possible physiological role of leptin in newborns through consumption of milk.

Chantier qualité GA; The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a decrease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT-PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells.

Chantier qualité GA; Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

In several species, placenta has been found to express GH-related proteins. In the ovine placenta, such a protein, ovine chorionic somatommamotropin, has been described, but its involvement in the fetal/placental growth process is not clearly established. The aim of this study was to investigate the occurrence of another GH-related peptide in the ovine placenta. Placental extracts (days 30-140 of pregnancy) showed GH immunoreactivity between days 35-70. SDS-PAGE analysis of these extracts indicated that this immunoreactivity corresponded to 22- and 28-kDa proteins. GH-like immunoreactivity was localized on cotyledonary frozen sections in the syncytium and the trophectoderm. Northern blot analysis of placental RNA showed the expression of GH-hybridizing transcripts migrating to the same position as that of GH pituitary messenger RNA (mRNA). Those transcripts were highly expressed between days 40 and 50. Their sequence analysis showed the existence of three GH mRNA (GHP1, GHP2, and GHP3). GHP1 is identical to pituitary GH mRNA and probably codes for the 22-kDa protein. GHP2 and GHP3 encode the same protein, which differs from GHP1 by four amino acids. This study establishes the expression of GH gene and GH-immunoreactive proteins in the ovine placenta.

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.

Objectives: Milk production in cows depends on all milk component synthesis and secretion by the mammary gland (protein, sugar, lipids…). This in turn involves a number of genes that are differentially expressed in this organ. In this study, we hypothesized that changes in gene expression profiles are related to differences in the methylation level at CpGs dinucleotides located within distal regulatory regions. As a first step, we focused on candidate regulatory regions of one casein gene (CSN1S1). Experimental methods: Holstein heifers were studied during early pregnancy (4 cows, day 60 of pregnancy) and lactation (8 cows, 7-10 weeks post-delivery). During lactation, the cows were studied over three periods. Period 1: twice daily milking for 5 days of both udders of each heifer. Period 2: differential milking for 7 days: right and left udders were milked twice and once a day, respectively. Period 3: both udders returned to twice daily milking. Milk yield was recorded every day over the three periods. Biopsies from left & right udders were taken at the end of period 2. Global DNA methylation was evaluated using the LUMA assay. DNA methylation of three CpGs located within a distal regulatory region CSN1 was analyzed using pyrosequencing after bisulfite treatment of genomic DNA. The mRNA and ncRNA expression levels have been quantified by RNASeq. Significance of results was studied with the Wilcoxon test. All experiments were conducted in compliance with French regulations on animal experimentation and with the authorization of the French ministry of Agriculture (Animal health and protection directorate, accreditation number 78-21). Results: During lactation when cows are milked twice daily, global DNA methylation in the mammary gland reaches 74.1%. The DNA methylation of each three CpG of a distal regulatory region of the CSN1S1 gene is low (33.1, 34.2 and 27.7%, respectively). In most cases (60%), the three CpG are simultaneously non methylated whereas only 17.8% are simultaneously methylated during lactation. In the once daily milking group, global DNA methylation is higher (75.9%) compared to the twice daily milking group but only significant if one of the eight heifers, which behaved differently from the others, was excluded (P=0.04). In the once daily milking group, the methylation level of each CpG is higher than that of twice daily milking group (P=0.02, P=0.07, and P=0.02, respectively). Furthermore, once daily milking induces inverse shifts with a decrease in simultaneaous non methylation (48%) and an increase in simultaneous methylation of the three CpG (26%). During pregnancy, the methylation level of each of these CpG is higher (57.1, 59.5, 50.1%, respectively). RNAseq data obtained from the mammary gland of those cows reveal the expression of ncRNA, specific to the mammary gland, in the vicinity of this region. Conclusion: A distal regulatory region of the CSN1S1 gene is hypomethylated during lactation as compared to pregnancy. Its methylation level increases during once daily milking and to a certain extent this corresponds to an increase in global DNA methylation state in the bovine mammary gland. These modifications may be related to transcription of the casein genes and to some ncRNA.

Le projet EpigRAni vise à caractériser la contribution de l'épigénétique à la variabilité phénotypique de caractères d'intérêt agronomique chez un mammifère d'élevage déjà hautement sélectionné, le bovin. L'épigénétique décrit la transmission des variations de structure de la chromatine qui régulent l'expression des gènes, sans modification de leurs séquences nucléotidiques. Ces variations participent à la variabilité des caractères d'élevage. L'identification et la mesure de variations épigénétiques offrent la possibilité de caractériser la dynamique de l'épigénome en vue de l'utiliser comme une nouvelle co-variable dans une perspective de génétique quantitative. C'est avec cet objectif qu'EpigRAni propose de tirer bénéfice de la disponibilité dans le laboratoire du partenaire 1, de plusieurs séries de bovins adultes génétiquement identiques, générés par transfert de noyaux de cellules somatiques (clones), animaux par ailleurs fertiles et parfaitement sains. Ces animaux expérimentaux produits sans recours à la recombinaison méïotique (génocopies) permettent de comparer les différences épigénétiques individuelles d'un même génome. L'ensemble de ces différences, ou certaines d'entre elles retenues comme cibles, peuvent alors être directement corrélées aux variations des phénotypes. Cette simplification expérimentale où le génome des animaux est "égal par ailleurs" constitue un avantage scientifique qu'Epigrani utilisera dans un premier temps pour mesurer la contribution de l'épigénétique à la variabilité phénotypique. Les analyses pan-génomiques de l'état de méthylation du génome sont conduites au cours de la différenciation et le développement de la glande mammaire et au cours du développement de l'unité foeto-placentaire impliqués réciproquement dans la production laitière et la fertilité, caractères d'intérêt agronomique majeur. Des régions-cibles du génome, les plus pertinentes quant à leur statut épigénétique, sont aussi utilisées chez des vaches laitières Holstein issues de reproduction naturelle et soumises à une traite différentielle. Ce projet propose de mettre en corrélation expression de gènes, statuts de méthylation et phénotypes de la glande mammaire ou phénotype de développement des tissus extraembryonnaires (défauts d'implantation associés à une réduction de leur fertilité). Projet de recherche académique, Epigrani est donc aussi une étude pilote visant à définir un nouvel outil -omique- pour accroître l'efficacité des nouveaux schémas de sélection génomique.

In the rabbit, alpha s1-casein is the major casein secreted in the milk. Transcription of the alpha s1-casein gene is induced by PRL. To define the positions of the cis-sequences involved in the control of rabbit alpha s1-casein gene expression by PRL, chimeric genes containing upstream regions of alpha s1-casein gene linked to the chloramphenicol acetyltransferase gene were cotransfected into Chinese hamster ovary cells with the plasmid expressing the rabbit mammary PRL receptor. It was observed that a distal fragment -3442/-3118 was responsible for a high induction of PRL sensitivity when linked in the 5'-position to a chimeric construct (-391/1774)-chloramphenicol acetyltransferase. A cooperation between distal and proximal regions of the alpha s1-casein gene is responsible for the PRL-dependent enhancer activity of the distal fragment. The mammary gland-specific nuclear factor-like binding sequence found around position -90 in the proximal promoter of the alpha s1-casein gene is involved in this cooperation. The distal fragment was further studied to determine the position of regulatory regions. A -3442/-3385 fragment was sufficient to induce a PRL sensitivity similar to that conferred by the larger -3442/-3118 distal fragment, but multiple interactions are likely to exist between other regulatory regions included in this distal fragment. Four DNA-binding regions (I-IV) have been identified within the reduced -3442/-3385 fragment by footprint experiments using rabbit mammary gland or liver nuclear extracts (NE). Protected area III is observed using both NE. Protected areas I, II, and IV are specific for lactating mammary gland NE. The sequences of areas I and IV share several homologies with the sequence of the mammary gland-specific nuclear factor-binding site.

National audience; Introduction : La glande mammaire subit de nombreux remodelages durant la gestation et la lactation mettant en œuvre de nombreux processus cellulaires (i.e. prolifération et différenciation cellulaire, apoptose...). Ces processus sont sous le contrôle de nombreux types de régulateurs, notamment les microARN (miRNA). Résultats: Après construction d’une banque de miRNA dans la glande mammaire ovine en début de gestation, nous avons cloné 54 nouveaux miRNA jamais décrits chez le mouton. Les séquences de ces miRNA sont très conservées par rapport à leur équivalent chez la vache, la souris ou l’homme. Ces résultats ont été complétés par une étude suivant l’expression des miRNA pendant le développement de la glande mammaire ovine adulte, en utilisant la technique de microarray. Nous avons montré qu’une centaine de miRNA est régulée suivant trois principaux profils d’expression: une baisse d’expression pendant la gestation et la lactation, un pic d’expression pendant la gestation ou une augmentation progressive d’expression à partir de la fin de gestation et au cours de la lactation. L’accumulation d’un miRNA représentatif de chacun de ces profils (respectivement miR-21, miR-205 et miR-200b) a été suivie par hybridation in situ à plusieurs stades du développement de la glande mammaire ovine. MiR-21 et miR-200b ont été détectés dans les cellules épithéliales luminales, alors que miR-205, quant à lui, l’a été dans des cellules basales pendant la première moitié de la gestation puis dans les cellules luminales pendant la deuxième moitié. Conclusion: Nos résultats décrivent une forte expression de miR-21 dans la glande mammaire pendant la gestation précoce, période correspondant à une prolifération cellulaire intense. Par ailleurs, nous montrons que miR-205 commence à être exprimé dans les cellules luminales pendant la seconde moitié de la gestation, alors que miR-200b est déjà présent dans ces cellules. Ces deux miRNA pourraient ainsi coopérer, comme cela a déjà été décrit dans une lignée cellulaire épithéliale (MDCK), notamment afin de maintenir le statut épithélial de ces cellules en réprimant un programme comparable à l’EMT (transition epithélio-mésenchymateuse) de façon à atteindre puis préserver le phénotype sécrétoire des cellules épithéliales mammaires. Perspectives : Afin de déterminer l’implication de ces miRNA dans ces régulations, nous envisageons de bloquer l’action de ces miRNA dans un modèle de dédifférenciation cellulaire (acini mammaires de souris en culture in vitro), puis de suivre la morphologie de ces cellules ainsi que l’expression de marqueurs épithéliaux et mésenchymaux.

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Epidemiological and animal studies have suggested that alterations to hormonal and metabolic environments during puberty can induce adverse effects on both lactation and mammary tumorigenesis (de Assis et al., 2006). Obese women have an increased risk of failing to initiate successful breastfeeding and can experience a premature cessation of lactation (Riva et al., 1999; Rasmussen et al., 2004). The effects of obesity on lactogenesis were highlighted during early studies in the rat, where this pathological condition was shown to reduce the chances of a successful outcome regarding pregnancy and lactation (Rolls et al., 1984). More recent studies have suggested that impaired mammary gland development and lactogenesis are possible causes of lactation failures and the high mortality rates observed among the pups of obese dams. In cattle, evidence concerning the relationship between growth rate, mammary growth and milk yield has led to the conclusion that an increased growth rate due to high feeding levels before the onset of puberty may impair subsequent pubertal mammary development and milk potential (Sejrsen, et al., 1997). Moreover, high energy feeding decreases mammary epithelial cell proliferation in areas of active ductal expansion at puberty (Davis Rinker et al., 2008). We have further investigated the impact of obesity on mammary gland development. A rabbit model of dietinduced obesity was developed, by feeding female rabbits with a high fat/high sugar (OB) diet (+276% fat and +269% sugar when compared with the control (C) diet), from 8 weeks of age until mid-pregnancy (day 14 of pregnancy, around the age of 24 weeks). Body weight gain between 21 weeks of age and day 14 of pregnancy was significantly higher (+10%) in OB animals, and was mainly associated with the development of more adipose tissue. Mammary gland morphology was altered in the OB group. Alveolar structures had invaded the entire fat pad, whereas they were more clustered in the control group. Moreover, on day 14 of pregnancy, the mammary ducts were made up of a cell monolayer, were dilated and filled with dense products. Numerous microvillosities were located in the apical region of mammary epithelial cells. Electron microscopy analysis revealed that casein micelles were present in the lumen of alveolae. Immunohistochemical studies of HF mammary tissue revealed a more abundant accumulation of the major rabbit milk proteins, alphaS1-casein, kappa-casein and Whey Acidic Protein (WAP), in both the alveolar lumina and secretory ducts. Milk protein synthesis in mammary tissue extracts was quantified by Western blot analyses. A clear accumulation of milk proteins was observed in OB animals but was almost undetectable in the controls. As revealed by BodiPy staining, lipids had also accumulated in the lumens and ducts. Taken together, these analyses revealed an early secretory phenotype in the OB group. These results show that diet-induced obesity, beginning before puberty, alters mammary gland development at mid-pregnancy in the rabbit. On day 14, mammary tissue in obese animals displayed a morphological aspect and functional profile similar to that normally observed at the end of pregnancy. Preliminary data indicated that these abnormalities affecting mammary development are not associated with agalactia at term, but that milk quality is affected and may influence the predisposition of offspring to obesity.

National audience; Les avancées technologiques de la biologie génèrent actuellement le recueil en masse de données qui permettent de décrire plus finement les grandes fonctions du vivant. Ces données participent à une connaissance approfondie des caractéristiques (traits) de chaque individu, c'est-à-dire de son phénome, résultat de la régulation de l’expression de son génome. La caractérisation de chaque phénotype implique un traitement informatique des données qui a révélé l’urgence de disposer d’un langage standardisé définissant sans ambiguïté les traits auxquels pourront se référer des utilisateurs très variés (généticiens, physiologistes, biochimistes, producteurs…). L’élaboration de ce langage a recours aux ontologies, ensembles structurés de termes et concepts qui s’adressent à un champ défini d’informations organisées pour répondre aux besoins des utilisateurs. Aujourd’hui, aucune de ces ontologies n’est spécifiquement dédiée aux traits phénotypiques des animaux d’élevage : poissons, volailles, mammifères. Face à cet enjeu, l’INRA en collaboration avec l’USDA a mis en place un programme nommé ATOL (pour « Animal Trait Ontology for Livestock») visant à définir et à organiser les caractères phénotypiques des animaux d’élevage. Ces caractères sont ceux relatifs aux fonctions et aux productions d’intérêt associées : nutrition et efficacité alimentaire, croissance et production de viande, glande mammaire et production de lait, système de reproduction et oeuf et ceux intégrant des préoccupations sociétales : bien-être des animaux d’élevage dans leur environnement. Le domaine « glande mammaire et production de lait » comprend deux sous-parties : l’une sur la glande mammaire avec des traits correspondant à sa morphologie et sa physiologie, l’autre sur le lait et le colostrum avec des traits correspondant à la production, la composition, aux propriétés physiques et sensorielles. ATOL devrait aider à établir des relations génotype/phénotype plus précises et à associer un phénotype à chaque allèle, en cernant mieux la variabilité liée à la plasticité physiologique et aux marques épigénétiques induites par le contexte environnemental. Cette démarche devrait ouvrir alors la voie à une approche systémique prenant en compte l’animal dans son environnement, son fonctionnement et ses mécanismes de régulation et non plus seulement une approche multicritère de la sélection animale.

International audience; The nuclear organization of mammary epithelial cells has been shown to be sensitive to the three-dimensional microenvironment in several models of cultured cells. However, the relationships between the expression and position of genes have not often been explored in animal tissues. We therefore studied the localization of milk protein genes in the nuclei of luminal mammary epithelial cells during lactation as well as in two non- expressing cells, i.e., hepatocytes and the less differentiated embryonic fibroblasts. We compared the position of a cluster of co-regulated genes, encoding caseins (CSN), with that of the whey acidic protein (WAP) gene which is surrounded by genes displaying different expression profiles. We show that the position of the CSN cluster relative to various nuclear compartments is correlated with its activity. In luminal cells, the CSN cluster loops out from its chromosome territory and is positioned in the most euchromatic regions, and frequently associated with elongating RNA polymerase II-rich zones. In hepatocytes and embryonic fibroblasts, the cluster is found preferentially closer to the nuclear periphery. Interestingly, we had previously observed a very peripheral position of the CSN locus in the nuclei of HC11 mammary epithelial cells weakly expressing milk protein genes. We thus show that cultured cell lines are not fully representative of the nuclear organization of genes in a complex and highly organized tissue such as the mammary gland and propose that the spatial positioning of the locus is important to ensuring the optimum control of CSN gene activity observed in the mammary tissue.

The rabbit κ-casein cDNA was cloned and sequenced. One of the isolated clones included almost the entire 5′ end, while another clone corresponded to the 3′ end of the cDNA. No polyadenylation site was found and therefore this clone did not harbour the complete cDNA. The amino acid sequence of a full-length protein was deduced from the nucleotide sequence obtained for this partial cDNA. It revealed the presence of a chymosin cleavage site and five potential phosphorylation sites. Rabbit κ-casein was compared with those already described in other species. The rabbit sequence is closer to the ovine than to the mouse sequence. This result supports the idea that Lagomorpha are not closer to Rodentia than to Artiodactyla. The cDNA described above was used to study κ-casein gene expression in the rabbit mammary gland. This expression was induced primarily by prolactin in mammary gland organoids and was similar to αs1-casein gene expression in vivo. The κ-casein gene present in the casein gene locus is thus subject to the same regulation as the αs1-casein gene, although it has evolved from a fibrinogen gene.

National audience; La glande mammaire se développe de manière cyclique à partir de cellules souches et progénitrices, en réponse à une régulation hormonale complexe. Plusieurs articles ont souligné le rôle que pouvaient jouer les microARN à la fois dans le maintien et la maturation des cellules souches. L’organisation spatiale du noyau, qui participe à la régulation l’expression des gènes et change au cours de la différenciation, n’a pas encore été étudiée dans ces cellules. Chez la génisse, les cellules progénitrices de la glande mammaire peuvent être identifiées sur les coupes de tissu par coloration à la fuchsine basique. Nous avons mis au point la détection de microARN par hybridation in situ sur cryo-coupes à l’aide de sondes oligonucléotidiques modifiées. L’expression de plusieurs microARN, notamment miR-21 et miR-205, a été étudiée au cours du développement de la glande mammaire de la vache et de la brebis. Mir-21 est un microARN oncogénique (oncomir) fortement exprimé dans de nombreux cancers et notamment les cancers du sein. MiR-205 a été montré par plusieurs équipes comme étant fortement exprimé dans les progénitrices mammaires chez la souris. Parallèlement, nous avons utilisé l’immunofluorescence sur cryo-coupes pour localiser les compartiments nucléaires, notamment les domaines d’hétérochromatine potentiellement impliqués dans l’épigénétique des cellules progénitrices. Nous avons montré qu’un simple marquage de l’ADN au DAPI permettait de distinguer les cellules progénitrices des cellules épithéliales en cours de différenciation, sur la base de la forme et de l’organisation interne des noyaux. L’identification des types cellulaires est optimale lorsque le tissu est inclus en paraffine. Les conditions d’hybridation in situ et d’immunofluorescence sont en cours d’optimisation afin d’examiner l’expression des microARN et l’organisation des noyaux à partir des coupes de tissu mammaire de génisse préparées avec cette méthode d’inclusion.

In many tissues, the features of cell nuclei are specific to their differentiated state, notably in terms of the nature and distribution of nuclear compartments and the position of chromosomes and genes. This spatial organization of the nucleus reveals domains that are differentially permissive for gene expression and may constitute an epigenetic mechanism that is involved in maintaining tissue-specific expression profiles. The mammary gland is a complex tissue in which mammary epithelial cells (MECs), which synthesize and secrete milk components, interact with other cell types (myoepithelial cells, adipocytes) and the extracellular matrix. MECs cultures can to some extent recreate cell differentiation in vitro and have been used to follow the development and functional importance of nuclear organization. They have made it possible to show how hormonal stimulation can lead to a remodeling of nuclear domains and the repositioning of genes specific to the mammary gland, such as milk protein genes. By modulating the growth conditions of culture in order to replace cells in a microenvironment similar to that of mammary gland tissue, it should be possible to study the role of this cellular microenvironment in nuclear organization.

International audience; During the development of tissues, complex programs take place to reach terminally differentiated states with specific gene expression profiles. Epigenetic regulations such as histone modifications and chromatin condensation have been implicated in the short and long-term control of transcription. It has recently been shown that the 3D spatial organization of chromosomes in the nucleus also plays a role in genome function. Indeed, the eukaryotic interphase nucleus contains sub-domains that are characterized by their enrichment in specific factors such as RNA Polymerase II, splicing machineries or heterochromatin proteins which render portions of the genome differentially permissive to gene expression. The positioning of individual genes relative to these sub-domains is thought to participate in the control of gene expression as an epigenetic mechanism acting in the nuclear space. Here, we review what is known about the sub-nuclear organization of mammary epithelial cells in connection with gene expression and epigenetics. Throughout differentiation, global changes in nuclear architecture occur, notably with respect to heterochromatin distribution. The positions of mammary-specific genes relative to nuclear sub-compartments varies in response to hormonal stimulation. The contribution of tissue architecture to cell differentiation in the mammary gland is also seen at the level of nuclear organization, which is sensitive to microenvironmental stimuli such as extracellular matrix signaling. In addition, alterations in nuclear organization are concomitant with immortalization and carcinogenesis. Thus, the fate of cells appears to be controlled by complex pathways connecting external signal integration, gene expression, epigenetic modifications and chromatin organization in the nucleus.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear “compartments”. Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types., Author Summary Several reports suggest functional relationships within the spatial organization of the nucleus, gene regulation and cell differentiation. However, it still remains difficult to extract common rules, mostly because i) most data have been gathered on limited sets of nuclear elements and in nuclei outside their normal physiological environment, and ii) few three-dimensional (3D) quantitative measures have been performed. Thus, we questioned whether common nuclear organization principles exist in the animal and plant kingdoms. For that purpose, we investigated the 3D distribution of centromeres/chromocenters in five populations of animal and plant nuclei: rabbit embryos at 8-cell and blastocyst stages, rabbit mammary gland epithelial cells and Arabidopsis thaliana plantlets. We set up adapted procedures to segment confocal images and developed a new analytical methodology based on distances between positions within the nucleus and centromeres/chromocenters. We showed that in all systems, despite large differences in chromosome number (44 in rabbit; 10 in A. thaliana) and genome size (rabbit estimated size 2.77 Gbp; A. thaliana 125 Mbp), centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation. This suggests that, whatever their specific features, conserved rules govern the spatial distribution of genomes in nuclei of differentiated cells.

International audience; Most of the development and functional differentiation in the mammary gland occur after birth. Epigenetics is defined as the stable alterations in gene expression potential that arise during development and proliferation. Epigenetic changes are mediated at the biochemical level by the chromatin conformation initiated by DNA methylation, histone variants, post-translational modifications of histones, non-histone chromatin proteins, and non-coding RNAs. Epigenetics plays a key role in development. However, very little is known about its role in the developing mammary gland or how it might integrate the many signalling pathways involved in mammary gland development and function that have been discovered during the past few decades. An inverse relationship between marks of closed (DNA methylation) or open chromatin (DnaseI hypersensitivity, certain histone modifications) and milk protein gene expression has been documented. Recent studies have shown that during development and functional differentiation, both global and local chromatin changes occur. Locally, chromatin at distal regulatory elements and promoters of milk protein genes gains a more open conformation. Furthermore, changes occur both in looping between regulatory elements and attachment to nuclear matrix. These changes are induced by developmental signals and environmental conditions. Additionally, distinct epigenetic patterns have been identified in mammary gland stem and progenitor cell sub-populations. Together, these findings suggest that epigenetics plays a role in mammary development and function. With the new tools for epigenomics developed in recent years, we now can begin to establish a framework for the role of epigenetics in mammary gland development and disease.

In the post genome era, the focus has shifted to understanding the mechanisms that regulate the interpretation of the genetic code. “Epigenetics” as a research field is taking center stage. Epigenetics is a term which is now being used throughout the scientific community in different contexts from physiology to cell biology and genetics with diverse meanings inherited from the past [1]. The term “Epigenetic” was first coined in the nineteen forties by C.H. Waddington when he proposed the concept of the “Epigenetic landscape” for the process of cellular decision-making during development [2, 3]. He developed this concept by taking into account heterogeneous data from genetics, evolutionary biology, and embryology, providing a conceptual model of the interaction of genetic material with its surroundings to produce a phenotype. Since then, the concept of epigenetics has evolved. It is now often defined as “the manifestation of a phenotype, which can be transmitted to the next generation of cells or individual, without alterations to the DNA sequence (genotype)”. An epigenetic event is a cell’s or organism’s response to external queues, which become part of its developmental repertoire, modifying gene expression profiles without alterations to the underlying DNA sequences. Epigenetic states can be maintained through mitoses but also through meioses and be transmitted from one generation to the next. In the animal kingdom, examples of epigenetic events transmitted from one generation to the next are rare [4]. In rats [5] or mice [6], nutrition has been shown to affect the phenotype of the offspring. Variability in the mouse Agouti phenotype (itself related to obesity and predisposition to diabetes) [7] has been reported in a homogeneous genetic background. A few examples of epigenetic inheritance in humans are still under debate such as grandchild phenotype which would depend on grandmother’s or grandfathers food supply or smoking [8]. On the contrary several responses to environmental conditions such as nutrition or stress at early stages of life resulting in different phenotypes during adulthood have been reported. Animal cloning clearly established a maternal imprinting during fetal life which affects growth and health in adults. In humans, it has been shown that mothers exposed to starvation during pregnancy give birth to children who will be on average smaller as adults [9]. When exposed to endocrine disruptors, women will give birth to males which will exhibit severe sexual disorders [10]. After birth, during the first week of postnatal life, maternal care also influences neuroendocrine and behavioral stress responses throughout life [11]. The most spectacular example might be the different phenotypes of “Queen” or “worker” observed in honey bees, depending on the nutrition of the larvae [12]. Thus epigenetics can explain the phenotypic outcome of interactions between environment and genetics, but it can also be seen as the basis of cellular differentiation. In that sense, it explains that in a multi-cellular organism development generates a plethora of cell types with distinct functions and diverse but stable gene expression profiles despite the shared genotype (same DNA) of the cells. E. Devinoy (*) INRA, UR1196, Genomique et Physiologie de la Lactation, 78 352 Jouy en josas Cedex, France e-mail: eve.devinoy@jouy.inra.fr

We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5′ flanking sequence could potentially confer. in vivo, high level expression or reporter genes. Transgenic mice were generated expressing a variant of human α1-antitrypsin, which has inhibitory activity against plasma kallikrein under the control of a 17.6 kbp DNA fragment located upstream of the rabbit WAP gene. Up to 10 mg/ml of active and correctly processed recombinant protein were detected in mouse milk, thus suggesting that the far upstream DNA sequences from the rabbit WAP gene might be useful for engineering efficient protein production in the mammary glands or transgenic animals.

The complete nucleotide sequence of the entire rabbit alpha s1-casein-encoding gene Aslca and its flanking regions was determined. These data represent the first complete primary sequence of an Aslca gene. The gene consists of 19 exons spread over 16 kb. Highly conserved sequences were found between this gene and other casein-encoding genes mainly upstream from the gene from position -180 to -10. Several repeated interspersed elements of unknown function were also identified within introns.

Summary Transgenic mice expressing foreign genes specifically in their mammary glands have been obtained by several groups in the world. The mouse is generally considered as a good reference animal to evaluate the efficiency of gene constructs to be used in larger mammals for the preparation of the corresponding recombinant proteins at an industrial scale. The method described here shows that mammary glands from lactating mice separated from their pups for one day spontaneously released 1.5 ml milk when stored at O'C. The proteins of milk obtained by this method were essentially similar to those obtained after milking. Human growth hormone (hGH) gene under the control of the rabbit whey acidic (WAP) gene promoter was expressed at a high level in the milk of transgenic mice (4 mg/ml milk in the mice examined here). hGH was present in milk obtained after milking or after the incubation of the mammary glands at O'C. In both cases, the hormone was present in essentially similar concentration, undegraded and b...

National audience; Milk contains mammary epithelial cells that are shed into milk during the lactation process. The opportunity to use milk cell RNA preparations to study mammary physiology provides a significant improvement over the use of biopsy samples and allows easy and repetitive sampling without damaging mammary tissue. In order to determine whether milk purified epithelial cells can reveal the variation of mammary gene expression after changes in milking frequencies, mammary biopsies and milk epithelial cells were prepared from unilaterally once daily milked cows. Five Hostein cows producing 15.7 kg of milk in well balanced udder-halves were subjected to unilateral once- and twice-daily milkings for 8 days. On the last day of unilateral milking, milk was collected from both udder-halves to prepare epithelial cells by centrifugation and a specific purification using an anticytokeratin antibody bound to magnetic beads. Mammary biopsies were performed on both udder-halves after milk collection. RNA was extracted from both milk purified epithelial cells and mammary biopsies. A transcriptomic analysis using a 22k bovine oligonucleotide array was performed to compare the transcript profiles between biopsies from once- and twice-daily milked mammary glands. Data were normalized by a global Loess regression and analysed using a mixture model using the Anapuce R package. Statistical significant raw p-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure which controls the False Discovery Rate. This analysis showed a differential gene expression of 504 transcripts. We obtained 193 and 232 transcripts up and down regulated, respectively, by once-daily milking compared to twice-daily milking. These transcripts were mainly involved in three functional families: « milk synthesis»; « cell dynamics » and « molecular transports ». The two first families are consistent with previous data showing a depletion of mammary synthesis activity and an activation of cell death during once daily milking. However, in order to precise the implication of the third family, more research has to be initiated to investigate the regulations involving molecular transports. Quantitative real-time reverse transcription PCR (RT-qPCR) analysis confirmed the differential expression in mammary biopsies of 22 mRNA, among 29 selected. The expression of these 22 mRNA and 4 stable transcripts was analyzed by RT-qPCR in the milk purified epithelial cell samples. Twelve transcripts were similarly expressed in both milk purified epithelial cells and mammary biopsies. These transcripts were mostly involved in milk synthesis. Four transcripts on 22 were inversely regulated between epithelial cells and mammary biopsies and ten transcripts on 22 did not significantly vary under once daily milking in milk epithelial cells. These differences may result from the fact that the mammary gland contains other types of cells than the epithelial ones. We therefore confirm that milk purified epithelial cells are a valuable source of mammary transcripts to analyze the effect of milking frequency on milk synthesis. However for peculiar transcripts a difference in gene expression is observed between biopsies and milk purified epithelial cells.

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