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Cattle suffering from inflammatory infection display sickness and pain-related behaviours. As these behaviours may be transient and last only a few hours, one may miss them. The aim of this study was to assess the benefit of combining continuous monitoring of cow behaviour via collar-attached accelerometers with direct visual observations to detect sickness and pain-related behavioural responses after a systemic inflammatory challenge (intravenous lipopolysaccharide injection) in cows of two different ages, proven by clinical, physiological and blood parameters. Twelve cloned Holstein cows (six ‘old’ cows aged 10–15 years old and six ‘young’ cows aged 6 years old) were challenged and either directly observed at five time-points from just before the lipopolysaccharide injection up to 24 h post-injection (hpi) or continuously monitored using collar-attached accelerometers in either control or challenge situations. Direct observations identified specific sickness and pain behaviours (apathy, changes in facial expression and body posture, reduced motivation to feed) expressed partially at 3 hpi and fully at 6 hpi. These signs of sickness and pain behaviours then faded, and quicker for the young cows. Accelerometers detected changes in basic activities (low ingesting, low ruminating, high inactivity) and position (high time standing up) earlier and over a longer period of time than direct observations. The combination of sensors and direct observations improved the detection of behavioural signs of sickness and pain earlier on and over the whole study period, even when direct signs were weak especially in young cows. This system could provide great benefit for better earlier animal care.

Estrogens are steroid hormones produced by the aromatization of androgens by the aromatase enzyme, encoded by theCYP19A1gene. Although generally referred to as “female sex hormones”, estrogen is also produced in the adult testes of many mammals, including humans. To better understand the function of estrogens in the male, we used the rabbit model which is an important biomedical model. First, the expression ofCYP19A1was localized, demonstrating that testicular estrogens are produced by meiotic germ cells inside the seminiferous tubules. Next, the cells expressing ESR1 and ESR2 were identified, showing that estrogens could exert their function on post-meiotic germ cells in the tubules and play a role during sperm maturation, since ESR1 and ESR2 were detected in thecaudaepididymis. Then, CRISPR/Cas9CYP19A1−/−genetically modified rabbits were analyzed.CYP19A1−/−males showed decreased fertility with lower sperm count associated with hypo-spermatogenesis and lower spermatid number. Germ/sperm cell DNA methylation was unchanged, while sperm parameters were affected asCYP19A1−/−males exhibited reduced sperm motility associated with increased flagellar defects. In conclusion, testicular estrogens could be involved in the spermatocyte-spermatid transition in the testis, and in the acquisition of sperm motility in the epididymis.

Resume Objectifs Selon le concept des « origines developpementales de la sante et des maladies », l’obesite maternelle predispose la descendance aux maladies chroniques. Pour de multiples raisons, une perte de poids preconceptionnelle est recommandee aux femmes obeses. Cependant, les avantages et les potentiels inconvenients pour l’enfant a naitre sont encore mal etudies. Methodes Nous avons evalue le developpement fœtal et analyse l’expression de modificateurs epigenetiques et de genes metaboliques dans le foie et le placenta. Les descendants nes de mere « controle », « obese » ou « perte de poids » ont ete soumis apres le sevrage a un regime controle ou hyperlipidique. Nous avons suivi les parametres metaboliques et les comportements olfactifs jusqu’a l’âge adulte. Resultats Les fœtus de mere obese presentent une restriction de croissance et une alteration de l’expression de modificateurs epigenetiques de la voie d’acetylation des histones. Les phenotypes metaboliques et olfactifs (obesite, dyslipidemie, intolerance au glucose et faible motivation et capacite a trouver une recompense alimentaire) s’expliquent principalement par le regime post-sevrage, mais l’obesite maternelle a un effet notable de conditionnement dependant du sexe : les mâles nes de mere obese developpent une obesite accrue par rapport a leurs congeneres. La perte de poids preconceptionnelle normalise les phenotypes fœtaux et metaboliques decrits ci-dessus, mais certains genes restent differentiels. De plus, elle a des effets inattendus sur les performances olfactives : la sensibilite olfactive peripherique et la motivation induite par le jeune sont reduites. Conclusions Nos resultats confirment les avantages d’une perte de poids preconceptionnelle pour la sante metabolique des descendants, mais revelent de potentiels effets indesirables tels qu’observes pour le comportement olfactif.

Animal toxicological studies often fail to mimic the complexity of the human exposome, associating low doses, combined molecules and long-term exposure. Since the reproductive potential of a woman begins in the fetal ovary, the literature regarding the disruption of its reproductive health by environmental toxicants remains limited. Studies draw attention to follicle development, a major determinant for the quality of the oocyte, and the preimplantation embryo, as both of them are targets for epigenetic reprogramming. The “Folliculogenesis and Embryo Development EXPOsure to a mixture of toxicants: evaluation in the rabbit model” (FEDEXPO) project emerged from consideration of these limitations and aims to evaluate in the rabbit model the impacts of an exposure to a mixture of known and suspected endocrine disrupting chemicals (EDCs) during two specific windows, including folliculogenesis and preimplantation embryo development. The mixture combines eight environmental toxicants, namely perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), dichlorodiphenyldichloroethylene (DDE), hexachlorobenzene (HCB), β-hexachlorocyclohexane (β-HCH), 2,2′4,4′-tetrabromodiphenyl ether (BDE-47), di(2-ethylhexyl) phthalate (DEHP) and bisphenol S (BPS), at relevant exposure levels for reproductive-aged women based on biomonitoring data. The project will be organized in order to assess the consequences of this exposure on the ovarian function of the directly exposed F0 females and monitor the development and health of the F1 offspring from the preimplantation stage. Emphasis will be made on the reproductive health of the offspring. Lastly, this multigenerational study will also tackle potential mechanisms for the inheritance of health disruption via the oocyte or the preimplantation embryo.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55-59 and 69-71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, "regulation of Rho protein signal transduction", implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Several studies have demonstrated that overexposure to pesticides can reduce mammalian sperm quality, impairing male fertility. Chlorpyrifos (CPF), a widely used organophosphate pesticide, was shown to impair spermatogenesis by inducing the formation of highly reactive toxic intermediates. To gain further insight into the mechanisms underlying the cytotoxicity and genotoxicity of CPF, bovine spermatozoa were exposed in vitro to environmental CPF concentrations and the motility, in vitro fertilization rates, DNA fragmentation, chromatin alterations, and methylation patterns were assessed. Motility and in vitro fertilization rates were significantly reduced in spermatozoa exposed to CPF, while DNA fragmentation and putative chromatin deconstruction appeared to increase at higher pesticide concentrations. In situ hybridization was carried out with X and Y probes on sperm samples exposed to different CPF concentrations, and subsequent analysis highlighted a significant percentage of spermatozoa with a peculiar morphological malformation, in which a narrowing occurred at the level of the hybridization. Analysis of potential abnormalities in the methylation pattern of NESP55-GNAS and XIST promoters displayed no differentially methylated regions in GNAS promoter relative to the control, whereas spermatozoa exposed to 10 μg/mL CPF had increased methylation variance in one region of imprinted XIST promoter. Our results provide support that CPF can induce a genotoxic effect on spermatozoa, impairig their ability to fertilize and support preimplantation embryo development in vitro. These observations are worrying since altered levels of sporadic methylation in genes of male gametes may affect the success of reproduction and contribute to infertility. Environ. Mol. Mutagen. 60:85-95, 2019. © 2018 Wiley Periodicals, Inc.

International audience; Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.

According to the new paradigm of the Developpemental Origins of Health and Disease (DOHaD), the environmental factors to which an individual is exposed throughout his life can leave an epigenetic footprint on the genome. A crucial period is the early development, where the epigenome is particularly sensitive to the effects of the environment, and during which the individual builds up his health capital that will enable him to respond more or less well to the vagaries of life. The research challenge is to decipher the modes of action and the epigenetic mechanisms put into play by environmental factors that lead to increased disease susceptibility or resilience. The challenge for health is to translate these scientific discoveries into action through, among others, the establishment of preventive recommendations to slow down the growing incidence of non communicable diseases.

The existence of non-genetic and non-cultural mechanisms that transfer information on the memory of parental exposures to various environments, determining the reactivity of the following generations to their environments during their life, are of growing interest. Yet fundamental questions remain about the nature, the roles and relative importance of epigenetic marks and processes, non-coding RNAs, or other mechanisms, and their persistence over generations. A model incorporating the various transmission systems, their cross-talks and windows of susceptibility to the environment as a function of sex/gender of parent and offspring, has yet to be built.

Epigenetics : a reading of the genome through stable / temporary / inheritable modifications Epigenetics refers to molecular processes checking the reading of the genome. These processes, including DNA methylation, the histone post translational modifications, as well as small and long non coding ARN, constitute the epigenetic machinery and participate to nuclear architecture. Stable, flexible, and quickly variable, the epigenetic status of cell nucleus is dynamic. After the fertilization, the parental genomes undergo epigenetic reprogramming, orchestrating the gene expression essential to the embryonic development. During the embryo development, the waves of cellular differentiation involved in organogenesis, come along with the implementation of cell-specific epigenome. Its stability is insured by the heritability of the epigenetic marks through the mitosis (and meiosis). It provides the maintenance of cell function associated with the stability of gene expression profile. Flexibility and/or reversibility of these marks take place in answer to diverse environmental changes. The importance of these processes is suggested by fetal programming, underlined the impact of in utero life on adult health and diseases. In order to exemplify these concepts, we will analyze the role played by the epigenetic programming during the spermatogenesis. Thus, epigenetics acts such as cell memory and origin of diversity between the individuals., L’épigénétique se réfère à un ensemble de processus moléculaires contrôlant le fonctionnement du génome. Ces processus, mettant en jeu la méthylation de l’ADN, les modifications post-traductionnelles des histones, de petits et grands ARN non codants, constituent la machinerie épigénétique et participent à l’architecture nucléaire. Stable mais aussi flexible, et variant rapidement, le statut epigénétique du noyau cellulaire est dynamique. Dès la fécondation, les patrimoines génétiques parentaux subissent une reprogrammation épigénétique orchestrant l’expression génique indispensable au développement embryonnaire. Au cours du développement, les vagues de différenciation cellulaire, nécessaires à l’organogénèse, s’accompagnent de la mise en place d’épigénomes cellulaires spécifiques dont la stabilité est assurée par l’héritabilité des marques épigénétiques à travers la mitose (voire la méiose). Ceci garantit le maintien de la fonction cellulaire de par la stabilité du profil de gènes exprimés. Flexibilité et/ou réversibilité de ces marques peuvent intervenir en réponse à divers changements environnementaux. Cette caractéristique prend toute sa dimension dans les processus de programmation foetale, soulignant l’empreinte de la vie in utero sur le devenir de la descendance. Afin d’illustrer l’ensemble de ces notions, nous prendrons divers exemples de biologie du développement avec une attention particulière concernant la place de la programmation épigénétique au cours de la spermatogenèse. L’épigénétique peut être considérée comme mémoire cellulaire et source de diversité entre les individus., Jammes Hélène. Épigénétique : une lecture du génome via des modifications stables / transitoires et transmissibles . In: Bulletin de l'Académie Vétérinaire de France tome 167 n°2, 2014. pp. 109-118.

SUMMARY We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses. Mol. Reprod. Dev. 80: 977–987, 2013. © 2013 Wiley Periodicals, Inc.

Daniel Le Bourhis, present adress: ALLICE, Nouzilly, France Sandrine Balzergue, present adress: IRHS, INRA, AGROCAMPUS-Ouest, Université d'Angers, SFR 4207 QUASAV; International audience; Cloning enables the generation of both clinically normal and pathological individuals from the same donor cells, and may therefore be a DNA sequence-independent driver of phenotypic variability. We took advantage of cattle clones with identical genotypes but different developmental abilities to investigate the role of epigenetic factors in perinatal mortality, a complex trait with increasing prevalence in dairy cattle. We studied livers from pathological clones dying during the perinatal period, clinically normal adult clones with the same genotypes as perinatal clones and conventional age-matched controls. The livers from deceased perinatal clones displayed histological lesions, modifications to quantitative histomorphometric and metabolic parameters such as glycogen storage and fatty acid composition, and an absence of birth-induced maturation. In a genome-wide epigenetic analysis, we identified DNA methylation patterns underlying these phenotypic alterations and targeting genes relevant to liver metabolism, including the type 2 diabetes gene TCF7L2. The adult clones were devoid of major phenotypic and epigenetic abnormalities in the liver, ruling out the effects of genotype on the phenotype observed. These results thus provide the first demonstration of a genome-wide association between DNA methylation and perinatal mortality in cattle, and highlight epigenetics as a driving force for phenotypic variability in farmed animals.

Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bovine spermatozoa is still scarce. In the context of genomic selection, more information on the epigenetic features transferred to the embryo alongside the paternal genetic heritage is necessary, in order to improve semen quality control procedures and bull fertility. We characterized the bull sperm methylome relative to bovine fibroblasts and monocytes using reduced restriction bisulfite sequencing (RRBS). A wide majority of differentially methylated cytosines (DMCs) were less methylated in sperm than in somatic cells. Consistent with previous studies in other species, these hypomethylated DMCs were enriched for genes relevant to the germline differentiation program and sperm functions. The most remarkable observation was a dramatic enrichment for repeats and particularly for satellites, which may partly account for the lower global methylation we observed in bull sperm relative to sperm from other species (LUminometric Methylation Assay). We next investigated whether fertility biomarkers could be identified in the sperm methylome. Based on field fertility indicators, we compared fertile ejaculates, subfertile ejaculates and ejaculates from bulls with a disappointing career in contradiction with genomic predictions (Montbéliarde breed, n=10/group). The median age at production was consistent among groups and the phenotypic characterization revealed no significant differences except for the proportion of motile sperm, which was reduced in disappointing bulls. We identified 4823 DMCs targeting genes involved in signaling pathways, angiogenesis and the response to growth factors. Since DNA methylation is affected by the CpG content of the genome and its alteration by DNA polymorphism, an integrative approach was conducted to identify CpGs and SNPs in correlation, which revealed both direct and indirect interactions between the genome and the epigenome.

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.

Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized- and nuclear transfer-EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT-EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT-EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast.

Children conceived through assisted reproductive technologies (ART) now account for a noteworthy proportion (-2.4%) of births in France. Considerable attention is being paid to the outcome of ART pregnancies. The vast majority of these children are apparently normal. However, they are at an increased risk of minor birth defects, low birth weight, and rare imprinting disorders such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS) and Silver Russel syndrome (SRS). Animal models are important for investigating the possible role of each step of ART (ovarian stimulation, gamete manipulation, in vitro fertilization, embryo culture and embryo transfer) in epigenetic reprogramming This review discusses these issues in the context of epigenetic and developmental abnormalities observed in animals following ART More research is needed on ART-induced errors, focusing not only on genomic imprinting but also on non-imprinted loci, which may help explain some of the more subtle longer-term health effects emerging from studies with animal models.

Resume Les modeles animaux montrent bien qu’un certain nombre de « stress precoces » tels que l’hyperglycemie maternelle (grossesses diabetiques), la sous-alimentation maternelle, l’obesite maternelle, la corticotherapie, l’insuffisance uteroplacentaire, l’hypoxie intra-uterine, conduisent a des adaptations metaboliques (notion de programmation ou empreinte, metabolique). Il existe un denominateur commun entre tous ces modeles d’etiologies tres differentes : lorsque les femelles de la generation F1 sont rendues gestantes, elles developpent une hyperglycemie gestationnelle et leur descendants (F2) sont intolerants au glucose. C’est ainsi que se met en place une veritable heritabilite de l’intolerance glucidique transmise de generation en generation. Cela suggere fortement qu’une hyperglycemie meme modeste, subie pendant la vie fœtale et/ou neonatale, contribue a la programmation du pancreas endocrine. Nous montrons qu’un tel scenario s’applique au rat GK/Par, modele de diabete de type 2 (DT2) spontane. En effet, a chaque generation, les meres GK/Par exposent leur descendance a une hyperglycemie moderee tout au long de la gestation et de l’allaitement. Nous proposons que l’hyperglycemie gestationnelle maternelle contribue a etablir, puis au cours des generations successives, a maintenir stable, l’heritabilite du phenotype « masse beta reduite », phenotype crucial dans la pathogenese du DT2. Des mecanismes epigenetiques pourraient rendre compte de l’empreinte pancreatique. Il est propose que des perturbateurs metaboliques intra-uterins puissent provoquer des changements transmissibles et cellule-specifiques de l’etat de methylation du promoteur et/ou de conformation de la chromatine (deacetylation des histones) de certains genes cruciaux pour la survie (Igf2, Igf1-R) et/ou la differenciation (Pdx1) des cellules beta. Cela conduirait a des changements perennes du niveau d’expression de ces genes.

Resume Des l’introduction des techniques d’Assistance medicale a la procreation (AMP), de nombreuses etudes ont ete menees pour evaluer leur impact sur la sante des enfants ainsi concus. La notion de risque epigenetique a ete evoquee et un lien entre AMP et pathologies associees a des anomalies de l’empreinte parentale a ete suggere : en particulier, dans le cas des syndromes de Beckwith-Wiedemann (BWS), d’Angelman (AS) et de Silver-Russell (SRS). Le cycle des marques epigenetiques (ecrasement dans les lignees germinales primordiales, etablissement de l’empreinte au cours des gametogeneses, lecture et maintien de l’empreinte dans les cellules somatiques) accompagne toutes les phases de la reproduction, la gametogenese, la maturation gametique, la fecondation et le developpement embryonnaire precoce. L’objectif de cette revue est de recenser toutes les informations issues de l’experimentation animale pouvant contribuer a une meilleure connaissance des perturbations de l’empreinte parentale, en liaison avec les differentes techniques utilisees en AMP et fournir des informations utiles dans la perspective d’ameliorer la prise en charge des couples engages dans un projet parental.

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.

International audience; The expression of the growth hormone receptor (GHR) gene was investigated in semitendinosus muscle during bovine foetal development in both normal and double-muscled Charolais foetuses which differ with respect to muscle development. Northern-blot analysis of foetal muscle RNA preparations with a GHR cDNA probe identified the 4.5 kb GHR mRNA as early as 130 days post-conception. In double-muscled animals, the expression of GHR mRNA increased from 130 to 210 days of gestation while it stayed stable in normal ones. It was significantly higher (P < 0.05) in double-muscled foetuses compared to normal ones from the second third of gestation. Northern-blot analysis of foetal muscle RNA preparations from both genotypes with a β-actin cDNA probe, revealed lower β-actin gene expression in double-muscled foetuses than in normal ones, suggesting a delay in the differentiation of muscle cells. In situ hybridisation revealed the localisation of specific GHR mRNA in muscle cells at all gestation stages analysed (130, 170, 210 days post-conception) but not in connective tissue surrounding the muscle cells. At the adult stage, the hybridisation signal was also very high and observed in muscle cells only. These results show the ontogeny of GHR mRNA in bovine muscle and demonstrate a difference between normal and double-muscled animals.

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50-60,000 Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50-60,000 Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.

The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.

Somatic cells are present in the milk throughout lactation and consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Our objective was to determine the efficacy of using somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. Over a 4-wk interval, cells were isolated from daily morning milk samples and samples taken 30 min after milking. They were characterized by direct cell counts and by flow cytometry analysis after immunostaining with antibodies directed against cytokeratin and CD45, a common leukocyte antigen. Epithelial cell counts within the morning milk ranged from 15 to 45% of total milk somatic cells. After-milking samples contained twice as many cells as did morning milk samples. The RNA was extracted from the somatic cells of both types of milk samples with equivalent efficiency (a mean of 1.2 microg RNA/mL of milk). Four mRNA variants of the alpha-S1 casein gene were detected by Northern blot analysis and the amount of each mRNA in milk cells was related to protein concentration in milk. The comparison between mRNA from the mammary gland and from congruently collected milk cells showed that relative amounts of mRNA for each milk-protein (alpha-S1 and kappa-casein and alactalbumin) were conserved. In a third experiment, daily milk cell RNA preparations were extracted to assess the effect of growth hormone (GH) on mammary gene expression; four goats were separated into two groups in order to perform a switch-back design consisting of three treatment weeks: Control, GH-Control or GH-Control-GH. In this study, treatment of goats with GH increased milk yields by 5%. Throughout the control and GH treatments, the expression of the three milk-protein genes studied were highly and significantly correlated (r = 0.949 and r = 0.958, P < 0.001 for, respectively, alpha-S1 and kappa-casein and for alpha-S1 casein and alpha-lactalbumin). During GH treatment, the three milk-protein mRNA abundances increased with the same pattern. In conclusion, the opportunity to use milk somatic cells for RNA preparation and analysis provides a significant improvement over the use of biopsy samples in assessing gene activity in the mammary gland and allows easy and repetitive sampling without damaging mammary tissue. Furthermore, we propose that this method could be used to investigate the transcriptional status of the mammary gland of an animal in relation to its genotype, nutritional and pathologic status, and under influence by hormonal factors.

Growth Hormone (GH), Insulin-like Growth Factors (IGFs) and IGF-Binding Proteins which modulate the IGFs' bioavailability (e.g. IGFBP-3, -4, -5), are essential regulators of bone remodeling. In this study, MG-63 human osteosarcoma cells were used as a model system to investigate the mechanism(s) whereby IGF-I and GH control IGFBP-3 gene expression. Physiological concentrations of IGF-I (1-20 nM) induced a dose-dependent increase in the steady-state amount of IGFBP-3 mRNA (maximal stimulation: approximately 9-10-fold). This increase was detectable 3 h after the onset of IGF-I treatment, was enhanced over a 24 h period, then plateaued until at least 30 h. Consistently, a dose-dependent increase in IGFBP-3 secretion ( approximately 40-50-fold at IGF-I concentrations/=16 nM) was observed by western ligand- and immuno-blot analysis of MG-63 cells conditioned medium, and its time course was similar to that observed for IGFBP-3 transcripts. IGFBP-3 mRNA stability (t(1/2) approximately 20 h) was identical in the presence or absence of IGF-I treatment. By contrast, human (h) GH treatment (24-72 h) of MG-63 cells did not increase IGFBP-3 secretion in the conditioned medium. Ectopic expression of recombinant rat GH-R resulted in hGH-enhanced expression of GH-responsive reporter gene constructs, but did not increase endogenous IGFBP-3 gene expression, suggesting that the GH unresponsiveness was not only due to the very low level of GH binding sites at the plasma membrane level. Altogether, these results support the conclusions that in MG-63 cells (i) transcriptional rather post-transcriptional mechanisms are involved in the IGF-I-induced increase of IGFBP-3; (ii) the abundance of GH-R is very low at the plasma membrane level; (iii) the dowstream GH-signaling cascade is fully functional in this human osteosarcoma cell line; and (iv) the endogenous IGFBP-3 gene is not responsive to hGH in human MG-63 osteosarcoma cells.

This study was designed to demonstrate the presence of epidermal growth factor (EGF) in the rat exorbital lacrimal gland. EGF precursor gene transcription was demonstrated first by RT-PCR analysis of lacrimal gland RNA using a set of specific primers and second by Northern blot analysis of rat lacrimal gland mRNA. A rabbit polyclonal antibody (rEGF2) directed against rat submaxillary gland EGF was used to detect EGF-containing proteins by RIA. Results indicate that the rat lacrimal gland does not contain detectable soluble and mature EGF but that the EGF immunoreactivity is associated with the membrane-enriched fraction. Analysis of the detergent-solubilized membrane proteins by gel filtration shows that membrane-associated EGF immunoreactivity was present as a high-molecular-mass protein. Moreover, as shown by Western blot analysis, a specific anti-rat EGF precursor antibody (ppEGF1) can immunoprecipitate a 152-kDa EGF-containing protein. Taken together, these results demonstrate for the first time both EGF precursor gene transcription and EGF precursor protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. The demonstration that EGF appears to be stored only as its full-length membrane precursor may provide important information to study the regulation of its secretory process.

International audience; Since the birth of the first somatic cell nuclear transfer (SCNT) animal in 1996, SCNT has been successfully applied to produce live animals in many species, but its successful application in animal production has been hampered by heavy fetal and postnatal losses and widespread public concern. So far, cattle remain the species with the largest number of cloned animals born. Success in animal cloning relies not only on the skill of the micromanipulator, but also on integrated knowledge and expertise in the pathophysiology and biotechnology of reproduction, cell and molecular biology, biochemistry, embryology, neonatology and obstetrics, image diagnostics, internal medicine, and surgical procedures, among other complementary disciplines and areas of knowledge. This chapter addresses the care of SCNT individuals and their recipients. The health and care in cattle will be described first, as most information is available in this species. Recommendations regarding pigs and other species will follow.

International audience; 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.

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/106 cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.

Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Znfinger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionnally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

L’empreinte parentale est un phenomene epigenetique complexe qui regule l’expression monoallelique de certains genes en fonction de l’origine parentale de l’allele. Les genes soumis a empreinte (GSE) participent au developpement placentaire et fœtal. Des defauts d’empreinte de certains GSE entrainent des syndromes cliniques associant une pathologie placentaire a l’origine notamment d’un defaut de croissance fœtale, et des manifestations neuro-developpementales. Environ 70 GSE ont ete identifies a ce jour chez l’homme. Nous avons developpe une strategie de criblage haut debit pour identifier de nouveaux GSE dans le placenta humain. Une premiere serie de validation a permis de confirmer le statut d’empreinte d’une dizaine de nouveaux genes [1] . Recemment, nous avons revele le statut de GSE pour le gene DSCAM (Down Syndrome Cellular Adhesion Molecule). Ce gene, qui se trouve dans la region critique du syndrome de Down, est ainsi le premier GSE identifie sur le chromosome 21. Notre etude montre que c’est l’allele paternel qui est exprime dans le placenta. Des resultats preliminaires nous laissent penser que l’empreinte de ce gene pourrait etre regulee par une region differentiellement methylee en amont du gene. L’etude par immunohistochimie de coupes placentaires montre une expression dans les cellules endotheliales et le syncytiotrophoblaste pouvant suggerer un role dans l’angiogenese placentaire via son ligand Netrin. Une etude est en cours a partir d’un modele cellulaire de trophoblaste avant et apres syncytialisation pour analyser son role dans ce type cellulaire. Par ailleurs, DSCAM est connu pour son role dans la mise en place des connections inter-neurones [2] . Une etude est en cours sur des modeles murins afin d’analyser le statut d’empreinte de ce gene dans le tissu nerveux et les consequences d’un defaut d’expression de son ligand Netrin sur le developpement placentaire.

Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors ( SERPINs ). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5 , and B7 presented significant differences in expression in ≥1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5 , and A8 presented complex methylation profiles. For SERPINA3 , the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.

Background As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes. Results Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of intra-uterine growth retardation (IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals. Conclusion We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5.10-5), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the “hypoxic” condition (2% O2), whereas apoptotic genes are overrepresented in the “normoxic” condition (20% O2). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O2tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.

Epigenetic marks, and especially DNA methylation, are at the interplay of both environmental and genetic factors. By facilitating the metabolic adaptation of highly selected rent animals to their environment, DNA methylation could contribute to the phenotypic differences observed between breeds. The aim of this study was to assess to which extent the methylome of 2 specialised cattle breeds – the dairy breed Holstein and the beef breed Japanese Black – could show some variability. We focused on the liver, which has a central role in metabolism and is therefore most susceptible to be affected by genetic and environmental variations. For each breed, both cloned and noncloned animals were included in the study. We used 9 adult Holstein cows aged from 5 to 15 years (5 healthy clones generated from ear skin fibroblasts of 4 genotypes, 2 cell donors obtained by AI and 2 other AI controls of unrelated genotypes, and 11 Japanese Black cows aged from 4 to 10 years (5 healthy clones generated from cumulus cells of one genotype and 6 AI controls of unrelated genotypes). The Holstein breed and Japanese Black breed were therefore represented by 6 and 7 genotypes, respectively. Liver samples were snap-frozen after slaughtering, and genomic DNA was extracted. To identify methylated regions, we used immunoprecipitation of methylated DNA followed by hybridization on a bovine promoter microarray (MeDIP-chip). The microarray targets the upstream region (–2000 to +1360 bp) of 21 416 genes (UMD3.1 assembly). After normalization of the data, enriched probes were identified using ChIPmix (Martin-Magniette et al. 2008). Results of exploratory analysis, including correlation clustering and principal component analysis, show a clear separation between the two breeds. A statistical test based on differences in the proportion of the enriched probes was used to identify differentially methylated regions (DMR) related to cloning and breed (Spatstat R package; http://www.spatstat.org/spatstat/). Only a restricted number of cloning-related DMR could be found (240). Interestingly, most of these DMR showed no overlap between Holstein and Japanese Black animals, maybe reflecting the different origin of the somatic cells used for cloning (fibroblasts v. cumulus cells). In contrast, we identified an important number of breed-related DMR (3642). These DMR were significantly enriched in genes involved in placental development and lactation, suggesting an adaptation of the two breeds to the different metabolic demand during gestation. Whether these epigenetic differences rely on environmental variations or genetic polymorphism remains to be elucidated.Research was supported by grants ANR-09-GENM-012-01 and Egide-JSPS Sakura 2012.

The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.

The lacrimal gland produces the complex aqueous portion of tears that contains many components including electrolytes and proteins.1,2 The pH, electrolyte concentration, and protein composition of lacrimal fluids are crucial to maintain the health of the ocular surface. The proteins that lacrimal glands synthesize and secrete are very specific, but only a few of them have been characterized. These proteins are thought to be involved in the bacteriostatic action of tears. It has been shown that human3,4 and mouse5 tears contain epidermal growth factor (EGF) or an EGF-like immunoreactivity. The EGF precursor mRNA has been detected by Northern blot in mouse6 and by RT-PCR in human7 lacrimal glands. Moreover, EGF-like immunoreactivities have been detected in mouse,6 rabbit,8 and rat9,10 lacrimal tissues, but the immunoreactive proteins have not been further characterized. The EGF receptor mRNA has also been detected, by RT-PCR, in the human lacrimal gland,11 and by RT-PCR and Northern blot in the rat exorbital lacrimal gland.12 The EGF receptor protein has been identified and shown to be activated by EGF in the rat tissue.12

To assess the precise role of IGF1 in benign and malignant breast diseases, we analysed the tissular localisation, characterised, and quantified specific insulin-like growth factor 1 (IGF1) binding sites in these heterogenous tissues, using histo-autoradiographic analysis (HAA). The 125I-IGF1 binding was performed on frozen tissue sections and analysed using 3H Ultrofilm autoradiography coupled to computerised image analysis. Competitive binding experiments using unlabelled IGF1, IGF2 and insulin showed that the tissues exhibited typical type I IGF binding sites. This specificity was confirmed by the use of alpha IR-3 monoclonal antibody, as inhibitor of 125I-IGF1 binding. IGF1 binding sites were detected in 18 human primary breast cancers, 12 benign breast tumours and two normal breast tissues. Using HAA we found that the human breast carcinomas studied exhibit a specific and high binding capacity for 125I-IGF1 exclusively localised on the proliferative epithelial component. The 125I-IGF1 binding activity of benign breast tumours or normal breast tissue was significantly lower than in cancerous tissues. There was a significant correlation between IGF1-R concentrations detected with HAA and those detected with a classical biochemical method. Moreover, HAA could be useful in further detailing whether a tumour is IGF1-R positive or negative HAA appears to be a useful method for the detection of growth factor receptors, specially in small biopsy specimens. Images Figure 2 Figure 3

The present report describes the first characterization of growth hormone (GH) receptor (GH-R) mRNA in the rabbit mammary gland. Northern blot analysis of poly(A)+ RNA isolated from several tissues of rabbit probed with a rabbit liver GH-R cDNA fragment revealed hybridization to only one transcript of 4.2 kb. A specific hybridizing signal appears in the mammary gland mRNA during gestation, when three different probes derived from liver GH-R cDNA and encoding respectively for extracellular, transmembrane and intracellular regions, were used. The signal is lower than in the liver but highly significant. These results indicate that the three regions are present and well conserved in the GH-R transcript found in the mammary gland. By S1 nuclease mapping analysis we demonstrated that the extracellular and transmembrane domains of mammary gland GH-R mRNA are strongly homologous to the liver GH-R mRNA. In addition, mammary gland GH-R mRNA is probably generated by mammary epithelial cells as demonstrated by the hybridization signal obtained using mRNA extracted from purified acini. The increase in the concentration of GH-R mRNA occurs during epithelial cell proliferation associated with a decrease in the proportion of adipocytes and connective cells at late gestation. The 4.2 kb GH-R mRNA species was also detected in ovine and porcine mammary glands during gestation, suggesting a probable expression of the related form of GH-R in these species.

DNA methylation of cytosines (5-mC) plays a crucial role in the epigenetic regulation of gene expression throughout development. The pattern of 5-mC is accurately established by de novo DNA methyltransferases (DNMT3a-3b) and inherited through mitosis by a maintenance DNA methyltransferase (DNMT1). Recently, another DNA modification, 5-hydroxymethylcytosine (5-hmC), was reported. 5-hmC is generated from 5-mC by TET proteins, and could be an intermediate in demethylation pathways. This study aims to determine the tissue- and cell- content of the 5-mC and 5-hmC amount by different approaches in mouse and in bovine species. Given the potential role of 5-hmC in nuclear reprogramming, it is crucial to obtain data in species with longer developmental time courses than the mouse.

International audience

National audience; L’épigénome du spermatozoïde mature est unique. En effet, il joue un rôle fondamental dans l’acquisition des propriétés morphologiques et fonctionnelles du spermatozoïde sans être en lien direct avec une activité transcriptionnelle. D’autre part, alors qu’il est le fruit de remaniements épigénétiques ayant démarré dès la vie fœtale, sa destinée est d’être reprogrammé rapidement après la fécondation. Malgré la large diffusion de semence bovine sur le marché de l’insémination artificielle, il existe peu de données sur la méthylation de l’ADN dans le spermatozoïde bovin. Nous avons d’abord caractérisé le méthylome spermatique bovin en comparaison avec des cellules somatiques adultes, et montré que certaines familles de séquences répétées (satellites, rDNA) présentaient un taux de méthylation très faible dans les spermatozoïdes. La surreprésentation de ces séquences dans le génome bovin, ainsi que leur faible méthylation, pourraient expliquer pourquoi les spermatozoïdes bovins présentent un taux de méthylation global plus bas que les spermatozoïdes d’autres espèces de mammifères. Nous avons ensuite étudié les paramètres intrinsèque (race du taureau), physiologique (âge à la production de semence) et pathologique (anomalies morphologiques des spermatozoïdes) associés à des variations du méthylome spermatique. La race, qui se confond avec l’origine géographique des taureaux étudiés (Holstein, Montbéliarde, Normande, Charolais, Blanc Bleu Belge et Abondance), semble modeler le méthylome spermatique au-delà de la présence de polymorphismes de séquence inter-races. Des taureaux de races différentes pourraient donc transmettre à leurs descendants non seulement des patrimoines génétiques différents, mais également des patrimoines épigénétiques différents. De manière plus surprenante, nous avons mis en évidence un effet majeur de l’âge du taureau au moment de la collecte de semence dans toutes les races étudiées. Les variations du méthylome liées à l’âge semblent quantitativement plus importantes que les perturbations associées à des anomalies morphologiques causant la réforme des taureaux, ce qui pose question sur la collecte de semence à un âge de plus en plus précoce dans les races laitières. Ces données montrent que dans leur ensemble, les pratiques de sélection et d’élevage des reproducteurs, ainsi que de collecte de semence, ont un impact majeur sur le patrimoine épigénétique véhiculé par le spermatozoïde. Il semble légitime de s’interroger sur les répercussions potentielles que pourraient avoir ces variations du méthylome spermatique sur le phénotype de la descendance. La mise en place d’un « contrôle qualité épigénétique » systématique de la semence bovine devient plus que jamais une nécessité, en cette période d’intense restructuration du secteur et de forte évolution des pratiques.

International audience; High variability in bovine milk production depends both on genetic and environmental factors. Whereas genomic selection has contributed to greatly improve milk production, mechanisms underlying the influence of the environment are mostly unknow. They can be studied using cloned cows (obtained through nuclear transfer), presumed to have identical genomes. Nine Prim’Holstein cloned cows and nine non-cloned cows were produced at similar periods of the year, raised in similar conditions, and inseminated at the age of 18 months. Eight cloned and 7 non-cloned cows delivered. Body condition score at calving was lower (ΔBSC=-0.6) for cloned cows (P=0.03). During the first 200 days in milk (DIM), milk yield varied greatly among animals but was similar between the two groups, whereas milk fat and protein contents varied less in cloned cows (P=0.004 and 0.09). Around 67 DIM, fat, protein and lactose contents (P

Alors que la semence bovine est un produit largement diffusé sur le marché de l’insémination artificielle, il existe peu de données sur la méthylation de l’ADN dans le spermatozoïde bovin. Le méthylome spermatique est pourtant le fruit d’une reprogrammation ayant démarré dès la vie fœtale, et joue un rôle essentiel dans la compaction de la chromatine et la protection du patrimoine génétique paternel. Nous avons donc mené une étude multi-échelle de la méthylation de l’ADN dans le spermatozoïde bovin comparativement à d’autres tissus et espèces.Une analyse globale par LUMA (LUminometric Methylation Assay, voir Prézelin et al.) a d’abord révélé un taux de méthylation extrêmement faible dans les spermatozoïdes bovins (autour de 45%, contre 60-80% pour les tissus somatiques examinés). L’analyse des spermatozoïdes d’autres espèces (béliers, boucs, étalons, verrats, souris mâles et Homme) a montré que cette hypométhylation était une spécificité du spermatozoïde bovin, et qu’elle était indépendante de la race et de l’état frais ou congelé de la semence. Les sites ciblés par LUMA ont été annotés par rapport à différents éléments géniques, aux îlots CpG et aux éléments répétés dans les espèces étudiées, ne révélant aucune particularité du génome bovin qui pourrait fournir des éléments de compréhension. Pour déterminer les séquences hypométhylées, nous avons ensuite comparé le méthylome spermatique avec celui d’autres tissus bovins (fibroblastes, foie, monocytes) en utilisant deux approches pan-génomiques couvrant des régions différentes du génome : RRBS (Reduced Representation Bisulfite Sequencing, voir Perrier et al.) et MeDIP-chip (immunoprécipitation de l’ADN méthylé et hybridation sur une puce de promoteur). Dans les deux cas, un nombre important de régions différenciellement méthylées (DMR, MeDIP) ou de CpG différenciellement méthylés (DMC, RRBS) ont été mis en évidence, dont la grande majorité se trouve être hypométhylée spécifiquement dans les spermatozoïdes (1144 sur 1678 DMR et 137861 sur 174103 DMC). Ces DMR/DMC hypométhylés sont enrichis en gènes de la spermatogenèse et du spliceosome ainsi qu’en séquences répétées de type satellite. Les résultats obtenus ont été validés par pyroséquençage. Si l’hypométhylation du spermatozoïde a occasionnellement été décrite chez l’Homme ou la souris, ou même chez le bovin pour les séquences satellites, l’originalité de ce travail est de montrer que cette hypométhylation est largement plus importante chez le bovin, et d’identifier les séquences hypométhylées en utilisant des approches complémentaires. Ces résultats suggèrent que des mécanismes spécifiques à l’espèce bovine sont à l’œuvre pour la reprogrammation ou le maintien de la méthylation dans la lignée germinale mâle.

National audience; Maternal obesity impacts fetal growth and pregnancy outcomes. Epigenetic regulation, that stably modifies the phenotype through chemical changes within the chromatin without alteration in DNA sequence, is one of the potential mechanisms to memorize the parental environment, leading to disease in adulthood. To counteract the deleterious effects of obesity on fertility and pregnancy issues, preconceptional weight loss is recommencled to obese women. Whether this weight loss is beneficial or detrimental to the offspring remains poorly explored. Epigenetic mechanisms could be affected by maternal weight changes, perturbing expression of key devel­ opmental genes in the placenta or the fetus. Our aim was to investigate the effects of chronic maternal obesity on feto-placental growth at term of gestation and in adulthood, along with the underlying epigenetic mechanisms. We also testecl whether preconceptional weight loss could· alleviate these effects. Female mice were fed either a control diet (CTRL group), a high-fat diet (obese group, OB) for 4 months, or a high-fat diet switched to a control diet 2 months before conception (weight loss, WL) group). At mating, OB females exhibited an obese phenotype while WL females normalizecl their weight and metabolic parameters. At embryonic day 18.5, fetuses from OB females showed fetal growth restriction and the odd-ratio for small for gestational age phenotype (SGA) was 3.2. These parameters were normalized for WL fetuses. The expression of 60 epigenetic machinery genes and 32 metabolic genes was measured in the fetal liver, placental labyrinth and junctional zone. One third of the epigenetic machinery genes were differentially expressed between at least two maternal groups, especially the histone acetylation pathway. Maternal weight loss normalized the expression of only a subset of these genes. A second cohort of offspring was followed from birth until 6 months for weight gain, food intake, glucose metabolism and cholesterol. During lactation, OB offspring rapidly caught up in weight and after weaning the diet-induced obesity was enhanced in OB males compared to CTRL. No obvious phenotype was observed in female offspring. Maternal weight loss rescued this phenotype. These results show that expression of epigenetic machinery genes and in particular histone acety­ lation regulators, is highly sensitive to preconceptional maternal metabolic features. The obesity­ induced transcriptional changes could alter the placental and hepatic epigenomes, contributing to SGA phenotype. Preconceptional weight loss alleviates the effects of obesity on fetal and adult weight but even if obesity was cured before conception, some effects were retained in the offspring phenotype at term. This study is an important step toward understanding immediate and long-term mechanisms linking maternal nutrition to fetal growth and adult health

We investigated the IGF-II gene expression in developing Semitendinosus muscle in foetal normal and double-muscled cattle. Samples from normal and double-muscled foetuses ranging from 90 to 210 d post-conception were collected and total RNA extracted. Northern blot analysis was performed using the human IGF-II cDNA probe. Five IGF-II transcripts, 5.1, 4.4, 3.7, 2.6 and 1.7 kb, were detected in muscle samples. Throughout gestation, all transcripts, except for the 5.1 kb one, decreased similarly in both genetic types. In double-muscled foetuses, the amount of the 5.1 kb transcript was higher than those of the other transcripts and its expression remained stable throughout the gestational stages analysed. These results indicated that the regulation of IGF-II gene transcription was distinct in both genetic types. The IGF-II foetal plasma concentrations increased throughout gestation. In bovine foetuses, the first muscle cell differentiation was concomitant with a high autocrine IGF-II mRNA expression and low plasma IGF-II levels in both genetic types. The second step of muscle cell differentiation was associated with high IGF-II plasma concentrations and the autocrine expression of IGF-II was reduced.

Background: Milking frequency influences milk production in cows by regulating milk synthesis and secretion (i). The higher or lower milk synthesis and secretion involve a number of genes that are differentially expressed. It has been well established that variation in DNA methylation at the CpGs located in the regulatory region of genes causes variation in gene expression. In this study, we hypothesized that milking frequency changes the milk yield through altered gene expression by modifying the methylation at CpGs dinucleotides located in gene regulatory regions. Materials & Methods: Eight heifers were studied in three periods. Period 1: twice daily milking for 5 days: both udders of each heifer were milked twice a day. Period 2: differential milking for 7 days: right udder was milked twice a day; left udder was milked once a day. 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 for histology, DNA, and RNA preparations. Global DNA methylation was evaluated using the LUMA assay. DNA methylation of three CpGs located within a distal regulatory region of one casein gene, CSN1, was analyzed using pyrosequencing of the PCR products amplified from bisulphite treated genomic DNA. The genome wide methylated regions will be captured and sequenced using MeDIP-Seq. The mRNA expression of CSN1 and other candidate genes obtained from MeDIP-Seq will be quantified using RT-qPCR. Results: Global methylation of the once daily milking group (75.9%) is higher compared to the twice daily milking group (74.6%). However the difference is not statistically significant (P=0.16). A closer look at the global methylation of each individual showed that one of eight heifers (8156) behaved differently from the others. When this individual was excluded, the two sets of data became significantly different (P=0.04). The DNA methylation of all three CpG1, CpG2, and CpG3 of once daily milking group is higher than that of twice daily milking group (P=0.02, P=0.07, and P=0.02, respectively). Those results are in accordance with global methylation profiles. Conclusion: Milking frequency modifies the DNA methylation to a certain extent at the global level and at the local CSN1 regulatory region in the bovine mammary gland. We are presently studying the relation of those modified DNA methylation and CSN1 mRNA expression using RT-qPCR. At the genome wide level, all possible methylated regulatory regions in the genome will be captured and sequenced using MEDIP-Seq. Gene expression of corresponding candidate genes will then be quantified. (i) Boutinaud et al. 2008. J Dairy Sci. 91: 988-98.

Chapitre 2; International audience; Since 2002, our INRA laboratory (Biologie du Développement et de la Reproduction) has developed a method to produce live somatic clones in rabbit, one of the mammalian species considered as difficult to clone. This chapter presents the technical protocol used nowadays to achieve the goal to obtain cloned embryos able to develop to term using fresh somatic cumulus cells.