A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific

peer-reviewedBackground: Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species.Background: Spermatozoa have a remarkable epigenResults: The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. Conclusions: These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis

Clonage chez le bovin, modèle de reprogrammation nucléaire : Effets épigénétiques dans les tissus extra-embryonnaires

Introduction : La méthylation de l’ADN, apposée directement sur les cytosines suivies d’uneguanine (CpG), est une des marques épigénétiques qui modulent l’expression des gènes sanschangement de la séquence nucléotidique et qui est transmissible au cours de la mitose et dela méiose. Après la fécondation, les pronucléus paternel et maternel subissent unereprogrammation avec un effacement des marques épigénétiques. Le noyau embryonnaire estalors le siège d’une apposition de nouvelles marques indispensables au programme dedéveloppement à réaliser. Le clonage par transfert d’un noyau somatique adulte dans unovocyte énucléé, nécessite une reprogrammation épigénétique conduite par la machinerieovocytaire. Ce processus est souvent l’objet d’erreurs plus ou moins importantes. L’efficacitédu clonage dépend de l’intensité de ces erreurs, qui dans certains cas conduisent à despathologies placentaires graves telles que le syndrome du gros veau. Ainsi le clonage peut êtreconsidéré comme modèle de perturbations épigénétiques dans un contexte génétique identique.Objectifs : 1) Déterminer le devenir du lignage trophoblastique en terme de méthylation del’ADN par un suivi des différents tissus extra-embryonnaires (trophoblaste, chorion,cotylédon, amnios, allantoïde) à 18, 40 et 60 jours de gestation. 2) Etudier les perturbations dela méthylation induite par la reprogrammation nucléaire dans ces tissus en comparant desfoetus obtenus par insémination artificielle (IA) et des foetus issus du clonage aux mêmesstades de gestation.Méthodes : Par « LUminometric Methylation Assay », nous avons déterminé la méthylationglobale de l’ADN génomique dans les différents tissus. Le différentiel entre la 5mC et la5hmC a été analysé par un test ELISA. L’étude de l’expression des gènes Dnmt et Tet a étéréalisée par RT-PCR quantitative.Résultats : Une augmentation graduelle du taux de méthylation au cours de l’évolution dulignage trophoblastique est observée, associée à des variations de l’expression des gènes Dnmt.Des différences de taux de méthylation entre tissus dérivés du trophoblaste sont significativeset conservées lors de croisement inter-races, suggérant une corrélation forte entre leméthylome et les fonctions de ces tissus. La 5mC et la 5hmC de ces 2 tissus ont été mesuréespar la méthode ELISA. La 5hmC est peu abondante. Le profil de la 5mC mesuré par LUMAest similaire à celui retrouvé par ELISA.Un différentiel de méthylation n’est observé entre IA femelles et clones que dans letrophoblaste à 18 jours de gestation (hyperméthylation des clones) et dans l’amnios à 60 joursde gestation (hypométhylation des clones). Systématiquement, les clones pathologiques sonthypométhylés par rapport aux clones non pathologiques.Conclusions : Les tissus issus du lignage trophoblastique, ainsi que l’amnios et l’allantoïde à60 jours sont de plus en plus méthylés pendant la gestation. L’hypothèse admise est que plusle tissu est méthylé et plus il est différencié afin de répondre à son rôle. Les cotylédons restenthypométhylés ayant de nombreuses fonctions à assurer. Les différences du taux deméthylation observées entre IA femelles et clones sont subtiles. Puisque l’embryon a réussi àsurvivre jusqu’au stade du prélèvement, cela suppose l’absence de perturbation majeure de laméthylation. Les pathologies placentaires sont certainement dues à des changements subtilsqu’une quantification globale ne peut détecter. Cibler les régions spécifiques d’unedérégulation pourrait être possible en mettant en oeuvre d’autres techniques d’analysepangénomique (Medip-Séquençage)

Obésité et perte de poids maternelles : programmation épigénétique dans le placenta et effets sur le phénotype des descendants adultes

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Hepatic global DNA Hypomethylation Phenotype in Rainbow Trout Fed Diets Varying in Carbohydrate to Protein Ratio

International audienceAbstract Background A high carbohydrate-low protein diet can induce hepatic global DNA hypomethylation in trout. The mechanisms remain unclear. Objective We aimed to investigate whether increase in dietary carbohydrates (dHC) or decrease in dietary proteins (dLP) can cause hepatic global DNA hypomethylation, and to explore the underlying mechanisms in trout. Methods Two feeding trials were conducted on juvenile males, both of which involved a 4-day fasting and 4-day refeeding protocol. In Trial 1, trout were fed either a high protein-no carbohydrate (HP-NC, protein 60% dry matter (DM), carbohydrates 0% DM) or a moderate protein-high carbohydrate (MP-HC, protein 40% DM, carbohydrates 30% DM) diet. In Trial 2, fish were fed either a moderate protein-no carbohydrate (MP-NC, protein 40% DM, carbohydrates 0% DM), a MP-HC (protein 40% DM, carbohydrates 30% DM), or a low protein-no carbohydrate (LP-NC, protein 20% DM, carbohydrates 0% DM) diet to separate the effects of dHC and dLP on the hepatic methylome. Global CmCGG methylation, DNA demethylation derivative levels, and mRNA expression of DNA (de)methylation-related genes were measured. Differences were tested by one-way ANOVA when data were normally distributed or by Kruskal-Wallis non-parametric test if not. Results In both trails, global CmCGG methylation levels remained unaffected, but the hepatic 5-mdC content decreased after refeeding (1–3%). The MP-HC group had 3.4-fold higher hepatic 5-hmdC and a similar 5-mdC level compared to the HP-NC group in Trial 1. Both MP-HC and LP-NC diets lowered the hepatic 5-mdC content (1–2%), but only the LP-NC group had a significantly lower 5-hmdC level (P < 0.01) compared with MP-NC group in Trail 2. Conclusions dHC and dLP independently induced hepatic global DNA demethylation in trout. The alterations in other methylation derivatives levels indicated the demethylation process was achieved through an active demethylation pathway and probably occurred at non-CmCGG sites

Exposition maternelle aux nanoparticules métalliques (Au-pvp, Ag, CeO2) par instillation: effets sur la fonction placentaire et le phénotype fœtal.

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Placental gene expression reveals reduced angiogenesis in primiparous mares

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Maternal obesity and weight loss: foeto-placental epigenetic programming and impact on adult offspring

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Nursing in the periconceptional period alters placental gene expression and subsequent foal growth

International audienceTo be profitable, equine breeders aim to produce a foal/mare/year, meaning that mares are bred while still nursing their previous foal. Most studies show that fertility as embryo/fetal mortality is increased in nursing mares. Long-term effects, however, have not been investigated. This project aimed to analyze the effect of nursing on placenta and foal growth. Multiparous Saddlebred mares aged 10-16 years were bred with the semen of one unique stallion on the 2nd heat after foaling (Nursing, N, n=6) or after having been left barren for 1 year (Barren, B, n=14). They were then managed as one herd in pasture and stalled individually in the same barn from around 6th month of gestation until foaling (11 months gestation). Placentas were recovered at foaling, measured and weighed. Stereology was performed using MercartorPro software on HES-stained paraffin embedded samples. Paired-end RNA-sequencing was performed on frozen placenta (Illumina, NextSeq500). Differential expression was analyzed (DESeq2) using a false discovery rate (FDR) <0.05 cutoff. Gene Set Enrichment Analysis was performed using KEGG, GO BP and REACTOME databases. Other data related to foal growth, metabolism and placenta were analyzed using a linear model with permutations using R software. A Frequently Sampled IV Glucose tolerance test was performed in mares at 300d of gestation and in foals at 6, 12 and 18 months of age. Lactation and foal growth were monitored until, respectively, weaning (≈6 months) and 18 months of age. N were more sensitive to insulin at 300 days of gestation (in median, 2.54 for N vs 0.78L/(mUI*min) for B, p<0.05). Gestation length was reduced (-8d) in N vs B. Although no morphological nor structural difference were observed, 38 genes were differentially expressed (23 and 15, respectively, over and under-expressed, FDR<0.05) in N placentas, among which 4 were directly involved in Wnt signaling pathway (WNT7, SFRP1 and LRP6 over-expressed and TRABD2A under-expressed in N placentas). Among the 226 perturbed pathways (183 GO BP, 16 KEGG and 27 REACTOME), only 13 were enriched in N placentas, mainly related with protein synthesis. Gene sets enriched in B placentas were involved in cell division and the regulation of inflammation and innate immunity. Although foal weight was similar at birth, post-natal growth was reduced in N, foals being lighter by 24kg at 18 months of age (p<0.05). No difference in carbohydrate metabolism was observed. Milk production, as analyzed through individual milking, was reduced at the end of the lactation period in N (N: 212.80 ±92.41 vs B: 401.62 ±100.64g, p<0.05) although milk quality was similar. In conclusion, nursing at the beginning of gestation is associated with reduced gestation length and functional placental adaptation probably compensating for the increased insulin sensitivity in N mares and enabling the birth of a normal weight foal. Reduced lactation potential, however, impaired long-term foal growth

Nursing in the periconceptional period alters placental gene expression and subsequent foal growth

International audienceTo be profitable, equine breeders aim to produce a foal/mare/year, meaning that mares are bred while still nursing their previous foal. Most studies show that fertility as embryo/fetal mortality is increased in nursing mares. Long-term effects, however, have not been investigated. This project aimed to analyze the effect of nursing on placenta and foal growth. Multiparous Saddlebred mares aged 10-16 years were bred with the semen of one unique stallion on the 2nd heat after foaling (Nursing, N, n=6) or after having been left barren for 1 year (Barren, B, n=14). They were then managed as one herd in pasture and stalled individually in the same barn from around 6th month of gestation until foaling (11 months gestation). Placentas were recovered at foaling, measured and weighed. Stereology was performed using MercartorPro software on HES-stained paraffin embedded samples. Paired-end RNA-sequencing was performed on frozen placenta (Illumina, NextSeq500). Differential expression was analyzed (DESeq2) using a false discovery rate (FDR) <0.05 cutoff. Gene Set Enrichment Analysis was performed using KEGG, GO BP and REACTOME databases. Other data related to foal growth, metabolism and placenta were analyzed using a linear model with permutations using R software. A Frequently Sampled IV Glucose tolerance test was performed in mares at 300d of gestation and in foals at 6, 12 and 18 months of age. Lactation and foal growth were monitored until, respectively, weaning (≈6 months) and 18 months of age. N were more sensitive to insulin at 300 days of gestation (in median, 2.54 for N vs 0.78L/(mUI*min) for B, p<0.05). Gestation length was reduced (-8d) in N vs B. Although no morphological nor structural difference were observed, 38 genes were differentially expressed (23 and 15, respectively, over and under-expressed, FDR<0.05) in N placentas, among which 4 were directly involved in Wnt signaling pathway (WNT7, SFRP1 and LRP6 over-expressed and TRABD2A under-expressed in N placentas). Among the 226 perturbed pathways (183 GO BP, 16 KEGG and 27 REACTOME), only 13 were enriched in N placentas, mainly related with protein synthesis. Gene sets enriched in B placentas were involved in cell division and the regulation of inflammation and innate immunity. Although foal weight was similar at birth, post-natal growth was reduced in N, foals being lighter by 24kg at 18 months of age (p<0.05). No difference in carbohydrate metabolism was observed. Milk production, as analyzed through individual milking, was reduced at the end of the lactation period in N (N: 212.80 ±92.41 vs B: 401.62 ±100.64g, p<0.05) although milk quality was similar. In conclusion, nursing at the beginning of gestation is associated with reduced gestation length and functional placental adaptation probably compensating for the increased insulin sensitivity in N mares and enabling the birth of a normal weight foal. Reduced lactation potential, however, impaired long-term foal growth