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International audience; DNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm. DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes. The DNA methylation dynamics during spermatogenesis has been previously described in mice and humans but nothing is known in productive livestock. However, these investigations in ruminants could be helpful to determine epigenetic events likely to be crucial for male fertility. Interestingly, a recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]. This result raises the question of the dynamic of DNA methylation in bovine male germline. We thus propose to define and compare the methylation dynamics of bovine and caprine germ cells during spermatogenesis. Analyses of 5mC immunohistochemistry were performed on adult testis sections to illustrate the presence of DNA methylation marks in various spermatogenic cells. As purification of the different spermatogenic populations is required to characterize DNA methylation more precisely, we firstly developed a flow cytometry-based method (Hoechtst-FACS) for cell sorting from goat testis. Fraction enrichments were evaluated using stage-specific markers by real time qPCR analyses. In addition, de novo DNA methyltransferase expression was studied on the different fractions of purified spermatogenic cells, giving some clues on DNA methylation dynamics. In the future, spermatogenic cells will be purified from both caprine and bovine testes, and together with gene expression analyses (RNA-sequencing), DNA methylation will be determined for each cell types by Reduced-Representation Bisulfite Sequencing (RRBS)

is a pdf file containing supplementary methods, supplementary references, eight supplementary tables and four supplementary figures. Table S1. reference genomes used for in silico analyses and origin of the files used for annotation. Table S2. in silico characterization of bovine reduced restriction (RR) genomes generated using different size selection criteria. Table S3. comparison of RR genomes obtained with a 40-290 bp selection size window in different species. Table S4. primers and PCR conditions used to generate the pyrosequencing templates. Table S5. pyrosequencing primers. Table S6. library characterization, mapping efficiency on the bovine genome (UMD3.1), coverage and average methylation in RRBS libraries. Table S7. results of comparisons between tissues by RRBS. Table S8. mapping efficiency on a Repbase artificial bovine genome, coverage and average methylation in RRBS libraries. Figure S1. Bootstrap analysis of global CCGG methylation in bull sperm from four different breeds. Figure S2. Genotyping of bull sperm and blood samples. Figure S3. Average methylation percentages for CpGs 5-500 and CpGs > 500 in each cell type, in reads uniquely aligned on a Repbase bovine artificial genome. Figure S4. Pyrosequencing of CpGs hypermethylated in sperm. (PDF 1840 kb)

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Nous avons développé un nouveau pipeline bioinformatique pour l’analyse du méthylome des animaux d’élevage à partir de données issues de RRBS (Reduced Representation Bisulfite Sequencing). Ce pipeline est le fruit d’un travail collaboratif étroit entre biologistes et bioinformaticiens de l’Unité et a permis de produire des résultats biologiques validés expérimentalement (voir Perrier et al. pour des exemples). Le pipeline peut dans un premier temps être exploité pour générer des génomes réduits in silico (« RR génomes ») en utilisant différents sites de restriction et des tailles de fragment variables. Un script original annote les CpGs couverts et détermine leurs caractéristiques génomiques ou leur présence dans des îlots CpG. Ces RR génomes permettent d’optimiser en amont le design expérimental en fonction du nombre de CpG couverts et de leur annotation. Après séquençage des banques RRBS, le logiciel Trim galore1 permet d’effectuer un contrôle qualité des séquences et de supprimer les adaptateurs de séquençage. L’alignement des séquences sur le génome de référence est réalisé à l’aide du logiciel Bismark2 qui prend en compte la conversion de l’ADN au bisulfite de sodium. Nous avons ensuite développé une série de scripts de contrôle qualité, comme le calcul de la distance entre deux reads issus d’un même fragment (en paired-end) ou la distribution de la couverture, ainsi qu’un script permettant de réaliser des analyses descriptives de manière intégrée. Pour des comparaisons impliquant des réplicats, les CpG différentiellement méthylés (DMC) sont identifiés à l’aide du package R MethylKit3 à partir des sorties du module d’extraction de méthylation de Bismark. Pour des comparaisons sans réplicats, nous avons conçu un script qui identifie les DMC « évidents » satisfaisant une différence de méthylation minimale entre les conditions. Un script a aussi été développé pour identifier les régions différentiellement méthylées (DMR) à partir de DMC voisines. Les DMC et les DMR sont ensuite annotées selon leurs caractéristiques génomiques et leur présence dans des îlots CpG, la distribution obtenue étant comparée avec celle de l’ensemble des CpG analysés afin d’identifier des enrichissements. Ce pipeline hautement paramétrable et facile à utiliser pour les biologistes est employé en routine au laboratoire pour analyser le méthylome de différents tissus bovins et de lapin4. Il peut également être appliqué à d’autres espèces ou adapté à des données de WGBS (Whole Genome Bisulfite Sequencing). Nous présentons ici des résultats obtenus à partir de données RRBS porcines publiées5. Ce pipeline offre donc une solution complète d’analyse de données de méthylation, de l’aide à la mise en place du design expérimental jusqu’à l’identification et la caractérisation de DMC et de DMR, et a été mis à disposition de la communauté FAANG

Le spermatozoïde a longtemps été considéré comme un taxi, protégeant et transportant son patrimoine génétique jusqu’à la délivrance dans l’ovocyte. Mais depuis quelques années de nouveaux arguments, autour des mécanismes épigénétiques, tels que les petits ARN non codants (sncRNA), proposent une nouvelle vision de la fonction du spermatozoïde par son implication dans le développement précoce de l’embryon (Gapp et al., 2014 ; Kotaja 2014 ; Boissonnas et al., 2013 ; Stopka et al., 2003 ; Liu et al., 2012). Or, chez les animaux de rente et en particulier chez les bovins, le contrôle de la fertilité est un élément central pour une diffusion optimale du progrès génétique. L’analyse des paramètres fonctionnels, traduisant la qualité du spermatozoïde, a longtemps été considérée comme une source importante de biomarqueurs du pouvoir fécondant de la semence. Mais malgré des résultats intéressants, la prédiction de la fertilité reste aujourd’hui inefficace (Sellem et al., 2015). C’est pourquoi la recherche de nouveaux biomarqueurs, tels que les sncRNA du spermatozoïde, a été entrepris. Notre étude a été conduite afin d’une part d’établir un catalogue exhaustif du contenu en sncRNA du spermatozoïde bovin et d’autre part de mettre en évidence de potentiels miRNA liés à la fertilité des taureaux. De l'ARN total a été extrait de 40 éjaculats provenant de taureaux Holstein et Montbeliard avec des fertilités contrastées, en utilisant un nouveau protocole amélioré. Deux contrôles qualité ont été effectués sur l'ARN extrait, afin de mesurer les quantités d'ARN (technologie Qubit) et de vérifier si un miARN de référence (miR125) a pu être détecté par RTqPCR. Les 40 banques NGS ont été préparées avec une sélection de taille (

International audience; Epigenetic modifications such as DNA methylation play a role in regulating gene expression and consequently in biological processes, such as those involved in health and disease. In dairy cows, profound changes occur during the post calving period, leading to immunosuppression and increased susceptibility to diseases. In order to monitor the postpartum health status of cows, one challenge is to describe the methylome of a purified subpopulation of immune cells, such as monocytes, and to determine its alterations/modifications in response to environmental or physiological changes. In this study, genome-wide DNA methylation profiles were obtained from purified monocytes (n=11), Peripheral Blood Mononuclear Cells (PBMC; n=4) sampled at D15 after calving and fibroblasts (n=2) using Reduced Representation Bisulfite Sequencing (RRBS). After sequencing and mapping to the reference genome (UMD3.1 assembly), 34.6% to 40.2% of uniquely mapped reads were obtained. Only CpGs covered between 10X and 500X (CpG10-500), were analyzed, which represented 55 ± 9.8 % of total CpGs with no significant differences between libraries. The chromosomic distribution of CpG10-500 was independent of the chromosome length (pvalue=0.48) but significantly associated with the coding gene content (pvalue=0.0001076). The global CpG10-500 methylation scores varied between cell types (fibroblasts: 47% ± 0.1, monocytes: 54.5% ± 2.9 and PBMC: 54.1% ± 1.8), and 19417 monocyte-specific Differentially Methylated Cytosines (DMCs) corresponding to 1572 DMRs were identified. This study therefore highlights CpGs and regions displaying a specific methylation pattern in monocytes, which could be targeted by epigenetic changes induced by environmental conditions (husbandry, nutrition, infection challenges…).

International audience; New sperm function in embryo development have emerged recently, relying on their small noncoding RNAs (sncRNAs) content. Indeed, involvement of sperm-borne miRNAs in mouse epigenetic inheritance has been evidenced and paternal sncRNAs have been shown to be dispensable for fertilization but crucial for the development of zygotes and 2 cells-embryos. Our study was conducted to unravel the sncRNA content from bull frozen sperm cells and identify miRNA associated with fertility. Total RNA was extracted from 40 ejaculates originating from Holstein and Montbeliard bulls with contrasting fertility, using a novel enhanced protocol. The two quality controls were done on RNA to measure RNAs quantities (Qubit technology) and check whether a reference miRNA (miR125) could be detected by RTqPCR. NGS sequencing libraries were prepared using small RNA (

International audience; The sperm cell has been generally considered as a cab driver, carrying and protecting his genetic heritage, but a new vision of its functions has emerged recently. Indeed, sperm cells also carry small noncoding RNAs (sncRNAs) into oocytes during fertilization. Although their roles remain largely unclear, involvement of sperm-borne miRNAs in mouse epigenetic inheritance has been evidenced and paternal miRNAs and/or endo-siRNAs have been shown to be dispensable for fertilization but crucial for the transcriptomic homeostasis and the developmental of zygotes and 2 cells-embryos. This study aimed at unraveling the sncRNA content from bull frozen sperm cells and identifying miRNA or piRNAs associated with fertility. Total RNA has been produced from 40 ejaculates originating from Holstein and Montbeliard bulls with contrasting fertility. To achieve this task, a novel protocol has been developed to ensure good quality and reproducibility. Quality control was ensured by total RNA quantification using the RNA HS Qubit Assay kit, as well as miR125 quantitation by RTqPCR (miRCURY LNA Universal RT microRNA PCR kit; Exiqon). Starting from 77 million of frozen sperm cells on average, RNA yield was 51 ± 15ng, leading to miR125 Cycle Threshold values in a 19 - 20 range. Only small RNA (

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.

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National audience; Selon le concept des origines développementales de la santé et des maladies, l'obésité maternelle prédispose la descendance à développer le syndrome métabolique à l'âge adulte. Une perte de poids préconceptionnelle est actuellement recommandée aux femmes obèses, mais ses effets pour la croissance du fœtus et pour la santé à l'âge adulte sont encore mal décrits. L'histoire pondérale maternelle pourrait être "mémorisée" par des mécanismes épigénétiques, perturbant l'expression de gènes clés du développement et du métabolisme et conditionnant ainsi à une susceptibilité accrue aux pathologies métaboliques. Nos travaux ont pour objectifs 1) d'étudier les effets de l'obésité maternelle sur le développement fœto-placentaire ainsi que les mécanismes épigénétiques sous-jacent 2) d'évaluer si la perte de poids préconceptionnelle entraine un bénéfice quant à ces effets ou si une mémoire de l'histoire pondérale maternelle est conservée dans la descendance. Nous avons développé un nouveau modèle murin : en période préconceptionnelle, des femelles ont été nourries pendant 4 mois avec un régime riche en lipides (HFD, pour high fat diet), entrainant une obésité (groupe OB), ou avec un régime contrôle (CD, pour control diet; groupe CTRL). Un troisième groupe a été nourri 2 mois avec le régime HFD puis 2 mois avec le régime CD, induisant une perte de poids (groupe WL pour weight loss). Nous avons le phénotype fœtal et placentaire au terme de la gestation et l'expression des gènes codants pour des modificateurs épigénétiques et des gènes impliqués dans le développement et le métabolisme, dans le foie fœtal, le labyrinthe et la zone jonctionnelle placentaires. Les fœtus OB présentent une restriction de croissance fœtale : ils sont "petits pour l'âge gestationnel" à terme. Ce phénotype est associé à une réponse transcriptionnelle dans le foie fœtal et le labyrinthe placentaire. Les gènes de la machinerie enzymatique impliquée dans l’acétylation des histones sont particulièrement affectés par l’obésité maternelle. La perte de poids a permis d’améliorer la croissance fœtale et l’expression génique, sans toutefois assurer une correction complète des effets de l’obésité préconceptionnelle. La machinerie épigénétique, en lien avec la restriction de croissance fœtale, semble donc particulièrement sensible au métabolisme maternel. Notre étude souligne l’importance de comprendre le rôle des régulateurs épigénétiques dans le contexte des pathologies métaboliques. Peu de preuves dans la littérature attestent les effets de l'intervention nutritionnelle chez les sujets obèses, chez l'homme et chez les rongeurs. Notre modèle nous permet d'apporter les premiers éléments sur les mécanismes du conditionnement développemental par la perte de poids préconceptionnelle maternelle.

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International audience; Spermatogenesis involves structural modifications including epigenetic remodeling of the genome. It allows the sperm genome to be compacted and protected, a prerequisite to the transmission of paternal genetic heritage to the next generation. Therefore, any environmental changes that impact epigenetic remodeling during spermatogenesis may alter sperm function and male fertility. DNA methylation is instrumental in this epigenetic remodeling, and alterations of methylation profiles have been associated with male infertility. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bovine sperm is scarce. The aim was to characterize the sperm methylome in cattle. The methods used were contrasted in terms of resolution (global rate vs base-level resolution) and genome coverage (genome-wide vs candidate regions). Global quantification by LUminometric Methylation Assay (LUMA) in six species revealed a species-specific behavior of DNA methylation in sperm. Methylation level was lower in bovine semen (~45%) than in other species (~70 to 80%), and this level was not affected by cryopreservation. Somatic cells showed similar level in all species. Breed-dependent variations were detected, suggesting genetic factors in the determination of methylation level. To map DNA methylation at a genome-wide scale, we used reduced representation bisulfite sequencing (RRBS) and Methylated-DNA Immunoprecipitation (MeDIP-chip). Differentially methylated regions between tissues (tDMRs) were identified when sperm, liver, fibroblasts and monocytes were compared. Some of these tDMRs were in the vicinity of genes enriched in important pathways for sperm physiology, and most of them were hypomethylated in sperm. Pyrosequencing validation of these tDMR is currently ongoing.

Description of the selection criteria of genes for the custom TLDA design and related bibliographic references.

Supplementary Figures S1 to S6. Food intake (FI) to body weight (BW) ratio in females during the preconceptional period. (a) P

International audience; 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.

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National audience; It is postulated that the developmental defects frequently observed in somalie clones arise from inappropriate reprogramming of the donor epigenome by the oocyte. We used cattle clones as a model to investigate the long­ lerm effects of nuclear reprogramming on both DNA methylation and the phenotype of a differentiated organ, the liver. Pathological perinatal clones and apparently normal adult clones, both produced within one decade, were studied, together with age-matched controls. We identified differentially methylated regions (DMRs) in gene promoters displaying variations according to age and to the clone status of the animals. Strikingly, whereas adult clones did not differ significantly from adult controls, perinatal clones showed a demethylation at DMRs inconsistent with their chronological age. Pathological features were found in the liver of perinatal clones only, with histological lesions and modifications to both histomorphometrical and metabolic parameters. The integration of DMRs with phenotypic datasets led to the identification of epigenetic alterations correlated to phenotypic abnormalities in perinatal clones. Genes important to the antioxidant response and energy metabolism were targeted by these alterations. For at !east one DMR, the aberrant methylation profile in perinatal clones was associated with altered gene expression. The liver defects encountered in pathological perinatal clones could be associated to the aberrant age of DNA methylation, at genes indicating potential deficits in oxidative metabolism. We propose that epigenetic ageing is an adaptation to the excessive oxidative stress induced by reprogramming that the pathological clones were not able to counteract during subsequent developmental stages.

International audience; Somatic cells bear a major potential as cell resource for biodiversity conservation: they carry the genome of both parents, and they can be collected whatever the age or sex of the donor. In fish, cryobanking of somatic cells is highly valuable because eggs and embryos cannot be cryopreserved. Then, the sole use of cryopreserved sperm for animal restoration requires long back-crossing with fresh eggs from plain strains. With somatic cells, the regeneration of the donor genotype will however rely on the nuclear transfer technology in which the donor cell nucleus is injected into a recipient oocyte. Although this restoration technology was developed in the 60’ for fish, the method is still far from being reliable enough for genome preservation and population restoration. Using the goldfish as a model, we showed that the most critical stage after nuclear transfer was the mid-blastula transition stage, when the 1000 cells embryo triggers its own genome activation. This strongly suggests that although the injected nucleus faced 10 rounds of mitosis, chromatin reprogramming was not reliable enough to allow the proper expression of the early development genes. We demonstrated that several marker genes whose promoter regions are hypomethylated in fertilized early embryos displayed various patterns of hypermethylation which were closer to the donor cell pattern. To some extent, the morphology of the embryo could be correlated to the level of the DNA methylation reprogramming. This demonstration reinforces the need to reprogram the donor cell prior to or during nuclear transfer, and the best strategies when facing fish cells and embryos will be discussed.

International audience; Epigenetic modifications such as DNA methylation play a role in regulating gene expression and consequently in biological processes, such as those involved in health and disease. In human, studies provide evidences that the methylome in peripheral blood mononuclear cells (PBMC) can reflect some disease susceptibility. In dairy cows, the post calving period is characterized by profound changes associated with an immunosuppression increasing the susceptibility to diseases. This study aims to quantify the global DNA methylation levels (dMe) by LUminometric Methylation Assay in bovine PBMC and in milk leukocytes and epithelial cells. Parity, milk production and quality, health and diet were reported from 52 Holstein housed at INRA’s experimental dairy farms. Genomic DNAs were extracted from 94 PBMC samples collected at D15 and D60 of lactation. PBMC-dMe varied from 72 to 86%. The proportion of different blood cell types were determined by cytometry and were found stable during the lactation period analyzed. dMe variations do not correlate with blood cell populations. No effect of lactation period, parity or breeding was observed. Moreover, dMe of PBMC was not significantly altered in pathological cows (mastitis or uterine post-partum disease, n=6) in comparison with healthy cows (n=18). For the 24 cows at the two lactation stages, milk leukocytes (ML) and epithelial cells (MEC) were also purified after milk centrifugation and immunomagnetic binding and gDNA extracted. Using the paired samples (PBMC-ML-MEC), dMe was found to be cell type specific: 77.08±1.8% in PBMC vs 65.6±4.1% in ML (significantly different, P

Session 16: discovery plenary session; Epigenetic databases in cattle ant the prediction of phenotypes from genotypes. 66. Annual Meeting of the European Federation of Animal Science (EAAP)

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International audience; DNA methylation (methylated (5-mC) and hydroxymethylated (5-hmC) DNA), is highly dynamic during mammalian embryogenesis. After fertilization, a loss of 5-mC of maternal (passive process) and paternal (active process) genomes takes place; 5-hmC accumulates in the paternal pronucleus and decreases progressively with the embryonic cleavage. In blastocyst, the first cell differentiation is associated with higher levels of 5-mC in the inner cell mass compared with the trophectoderm. During foeto-placental development, the patterns of 5-mC and 5-hmC are established according to cell specification and tissue differentiation. In some bovine blastocysts produced by somatic cell nuclear transfer (SCNT), a global DNA hypermethylation has been observed in the trophectoderm. Defaults of placental and foetal development are frequent after SCNT in mammals. Whether these pregnancy pathologies are associated with alterations of global DNA methylation remains to be analysed. In this study, the patterns of DNA methylation were determined in extra-embryonic and foetal tissues during pregnancies obtained by artificial insemination (AI) versus SCNT i) by a global quantification using a LUminometric Methylation Assay and ii) by immunohistochemistry with specific antibodies. Great variations in methylation levels between extra-embryonic and fœtal tissues at D60 of gestation were observed from 30% to 90% (chorionic villi > chorion > liver > heart > brain > amnion > allantois). Chorionic methylation content increased significantly from D18 until term of gestation. Effects of SCNT on trophoblast methylation level in blastocyst were conserved in trophoblast at D18 and D40 and in cotyledons at G60 only. However, by immunocytochemistry, only the mesenchymal part of chorionic villi was stained using 5-mC antibody and an unexpected 5-hmC staining was found in various placental and extra-embryonic cell types, suggesting a role of this epigenetic mark in all lineages in bovine.

International audience; Nuclear reprogramming: Methods and Protocols, Second Edition includes not only classic methods to perform nuclear transfer in different species but also several techniques to assess the early and late development of the reconstructed embryo, at the cellular, molecular, and epigenetic level. Over the past several years, many technical improvements have been made to improve somatic cell nuclear transfer (SCNT) efficiency, all of which are reflected in the detailed chapters of this fully revised collection. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Nuclear Reprogramming: Methods and protocols, Second Edition will be interest not only to cloners but also to researchers concerned with studying the development of embryos.

International audience; Epigenome is the essential mediator of the effect of environmental exposures on development. Epigenetic studies show that alterations in DNA methylation marks are associated with developmental reprogramming and linked to environmental exposures. Our objective is to develop different Intra Uterine Growth Restriction (IUGR) rabbit models and to study the alterations occurring in the methylome of the placental unit throughout gestation. The relevance of the rabbit model resides in its placentation similarity with humans. As little is known about rabbit epigenome, we are developing a strategy to establish a toolbox to study the rabbit methylome. First, studying the methylome of the placental unit will be performed by MeDIP-seq on pooled samples to generate a methylation overview in both placenta (fetal compartment) and decidua (maternal compartment) at term. In a second time, a microarray will be designed for individual analysis to establish early epigenetic events related to placenta from IUGR. The array design will integrate methylated sequences from MeDIP-seq data but also promoters of differentially expressed genes obtained by transcriptomic analysis performed on the same samples. Finally, to validate our results, methylated regions of interest will be studied by pyrosequencing after bisulfite treatment. The identification of critical epigenetic marks alterations associated with IUGR will allow a better understanding of the IUGR process and an identification of key actors in placental function. In future studies, these marks could be linked to the establishment of a specific phenotype at adulthood and tested as biomarkers at birth to define the risk of developing an adverse phenotype. In addition, this study will present the first overview of the rabbit methylome and lead to the development of epigenomic tools that can be used systematically.

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A global increase of the male infertlity is described for different species. Human infertility affects 10-15% of couples and among infertlity causes, one third has a male origin (Louis, 2013). The genetic causes of male infertility have been searched (ka1yotypic abnonnalities, microdeletions, mutations ; Maduro, 2003). ln cattle, the importance of male contribution to reproductive success is weil documented, bull breeding soundness evaluation are a commercial practice and there are specifie guidelines for semen evaluation. Hovewer, a surprisingly law number of quantitative trait loci (QTLs), genome-wide association studies (GWAS) and genomic selection (GS) approaches have reported candidate genes associated with fertility tmits (Fortes 2313). ln recent years, it become inc1*easingly ciear that epigenetic mechanisms are critical during spennatogenesis and that perturbations in these mechanisms can result in male sublinfeltility (Zamudio, 2008; CaiTell, 2012; Chalas­ Boissonnas, 2013). The current knowledge on epigenetic that occur during male meiosis is discussed in this review. A special attenton is focused on DNA methylation, histone tail modifications, targeted histone reteltion and protamine incorporation into the chromatin, and small and long non coding RNA production. Ali these epigenetic mechanims are involved in highly regulated processes such as silencing of gene and retrotransposon, chromatin condensation and packaging of DNA. We will discuss the importance of a better understanding of how epigenetic changes in the spenn may have a causative role in etiology of infertility. Perturbations in the establisment and lor maintenance of any of these epigenetic marks could be induced by environmental factors and could affect the fertility status. These epigenetic marks constitute also the paternal contribution to embryo development. Finally, the recent studies focused on t*ansgenerational and paternal effects in mice and in human are discussed.

Reprogramming the differentiated cell to totipotency can be obtained following the introduction of its nucleus into an enucleated oocyte, a procedure known as cloning. We are using cattle clones as a model to assess the inter-individual epigenetic variability and its consequences on phenotypes, including agronomical relevant traits and developmental pathologies. Indeed, the developmental defects frequently associated with cloning could be related to the insufficient extent of reprogramming, leading to perturbations of the nuclear microenvironment of the early embryo with long term consequences. To identify epigenomic patterns affected by incomplete reprogramming, we used immunoprecipitation of methylated DNA followed by hybridization on a new bovine promoter microarray (MeDIP-chip). The microarray targets the upstream region (-2000 to +1360bp) of 21416 genes (UMD3.1 assembly). We first focused on liver, because overgrowth of this organ is often observed in clones. The microarray has been hybridized with MeDIP samples from livers of normal Holstein animals (4 perinatal controls and 8 adults, obtained by artificial insemination and all healthy) and livers of Holstein clones (7 perinatal clones, either stillborn or suffering from severe pathologies, and 7 adult clones, with normal to pathological phenotypes). After normalization of the data, enriched probes were identified using ChIPmix (Martin-Magniette et al., 2008). Results of exploratory analysis, including correlation clustering, Principal Component Analysis (PCA) and Independent Component Analysis (ICA), will be presented. A statistical test based on differences in the spatial distribution of the enriched probes was used to identify Differentially Methylated Regions (DMR) related to age and cloning. In age-related DMR, we show that cloning erases the methylation signatures associated to age. Conversely, analysis of cloning-related DMR revealed that bovine clones behave like adults independently of their age.

National audience; Single layer centrifugation (SLC) with Androcoll-E™ enables a better sub-population ofspermatozoa to be selected from stallion ejaculates. Installions with fertility problems, SLC-treated sperm seems to result in normal pregnancy rates. Since no studies have been performed on the effect of Androcol!-ETM treatment on pregnancy rates obtained from fertile stallions, this study was designed to investigate whether SLC could be used to enbance fertility rates of normal stallion. Ejaculates obtained from a SIRE were used to study quality and longevity of the sperm recovered after centrifugation in Androcol!-ETM as weil as pregnancy rates obtained after AI with and without SLC-centrifuged sperm. The sperm's characteristics not recovered by SLC-treatment were also investigated and results were compared by ANOVA. SLC-recovered sperm presented the highest percentage oflive cells and membranes osmotically active (HOS test) while the highest percentage of sperm with high mitochondrial potential (il'JI mit; JC-1) and DNA integrity (SCD test) was retained in the upper layer. Sperm motility in SLC-selected samples cooled for 72 h didn't differ from non­ SLC-selected sperm. A crosstabs procedure (chi-square test, x2) was used and per cycle fertility obtained with Androcol!-ETM treated semen (78%, 9 mares; 7 cycles DG+) didn't differ (x2(2)00; P=0.527; n=18) from per cycle fertility obtained with non SLC-treated sperm (89%; 9 mares; 8 cycles DG+). In conclusion, our results suggest thal fertility is independent oftreatment with Androcol!-ETM. Answer to REviwers: We have no data with sub fertille stallions regarding in vivo and in vitro comparison.