| The effects of father-to-offspring transmission attract recent attention.Increasing evidences from Drosophila melanogaster,zebrafish,and mammals suggested that epigenetic information passed on by the sperm allows for the inheritance of information on paternal environmental exposure,especially the changes in paternal diets,including the modulation of fat,sugar,and protein.Mechanistically,altered DNA methylation,histone modifications,chromatin state,as well as changed expression of small non-coding RNAs,have been reported to mediate this transgenerational effect.Small non-coding RNAs(sncRNAs)regulate DNA methylation,histone modifications,and mRNA transcription,and can induce non-Mendelian transgenerational inheritance in mammals.Specially,altered sperm miRNA profiles have been observed following paternal exposure to diet.However,since sperm harbors a diversity of noncoding RNAs,the specific population of RNAs,such as long noncoding RNAs(lncRNAs),which mediate intergenerational epigenetic memory remained unknown.Similarly,lncRNAs could also regulate several epigenetic modifications,including DNA methylation,histone modification,small RNAs expression,which indicated that lncRNAs could further influence the gene expression by several roles.Meanwhile,changed lncRNAs expression were accompanied by environment changes.Herein,we hypothesized that the miRNAs and lncRNAs could show alterations in expression profile responds to different paternal diet and transmitted certain metabolic changes from father to offspring.By both affecting supply of S-adenosylmethionine(SAM)and the removal of S-AdenosylL-homocysteine(SAH),folate could serve as an important carrier of methyl groups in one carbon metabolism and further influence amino acids metabolism,nucleotide synthesis,epigenetic processes.In the present study,with the goal to study the transgenerational regulatory effects of paternal normal dietary supplement in birds,the folate were added into the feed of the breeder roosters.In the present study,using a chicken model that were fed with different concentration of folate throughout life,we addressed how paternal folate supplement exerts a transgenerational effect in birds,and we further studied the underlying mechanisms.We show that the paternal folate supplement will alter the differential expression of noncoding RNAs(miRNAs and lncRNAs)in sperm with consequences for altered pregnancy outcomes,changed blood and hepatic lipids metabolite and hepatic gene expression related to lipid metabolism and gluconeogenesis process of breeding cocks and their offspring broilers.Exp.1 Phenotypes and metabolic changes in breeding cocks and their offspring broilers induced by dietary folate supplement of breeding cocksIn the present study,the growth performance and the organ development of breeding cocks and their offspring chicks were recorded.F0-breeding cocks with extra chronic dietary folate supplements exhibited a reduced body weight,increased organ index including the liver and Bursa of Fabricius.Meanwhile,the duodenum morphology,including the villus height and the radio of the villus height to crypt depth,could be significantly increased with the extra folate supplement.Moreover,referring to the phenotypes changes of the offspring broilers,we found that the born weight of the 1-day-old broilers could be significantly increased and the feed conversion ratio during the whole 21 days and the period of 14-21 day were significantly decreased when we supplied extra folate to breeder cocks.Meanwhile,the organ index of Bursa of Fabricius of 1-d-old and 21-d-old offspring broilers could be also significantly increased in those four folate supplement groups.Furthermore,we also found that the intestinal development of the offspring broilers were also significantly changed.Compared with those broilers in control group,those indexes of the broilers in the folate supplemental group,including the relative cecum length of the 1-d-old offspring broilers,the V/C of duodenum in 21-d-old offspring broilers,the villus height and the V/C of the jejunum in the 1-d-old offspring broilers,were all significantly increased,which indicated that the paternal folate supplement was benefited to the intestinal development of offspring broilers.Altogether,these data indicated that the paternal folate supplements were benefited to the embryonic and postnatal development of their offspring.Considering the roles of folate in one-carbon metabolic process and its related roles in regulating lipid metabolism and glycometabolism process,we further measured a series of indexes of breeder roosters and their offspring broilers which could represent these related metabolic process.In plasma,a significantly increase of the total glyceride(TG)concentration and free fatty acid in 1-d-old offspring broilers,a significantly decrease of the high-density lipoproteincholesterol(HDL-C)concentration in breeder roosters and offspring broilers on 21-d-old,a significantly decrease of the low-density lipoproteincholesterol(LDL-C)in breeder roosters,and offspring broilers on both 1-d-old and 21-d-old,a significantly decrease of very low-density lipoproteincholesterol(VLDL-C)concentration in breeder roosters and offspring broilers on 1-d-old,as well as a significantly decrease of adiponectin concentration in breeder roosters and offspring broilers on 21-d-old were separately founded in the folate supplemental groups when compared with the breeder roosters and their offspring chicks in control group.Following,we further analyzed the lipid metabolic indexes of livers.Compared with the control group,a significantly increase of TG,HDL-C,and carnitine concentration were only detected in the breeder roosters from folate supplementary groups.However,a significantly increase of total cholesterol were founded in the breeder roosters,1-d-old offspring broilers,and 21-d-old offspring broilers of the folate supplementary groups.Meanwhile,compared with the control group,a significantly increase of FFA in breeder roosters and 21-d-old offspring chicks and a significantly increase of malate dehydrogenase(MDH)in 1-d-old offspring broilers of the folate supplementary groups were also founded.Furthermore,the hepatic glycometabolism and of breeder cocks and their offspring broilers were also recorded in the present study.As results,in folate supplementary group,the activities of phosphoenolpyruvate carboxykinase(PEPCK)in breeder rooster and 1-d-old offspring broilers,the activities of pyruvate kinase(PK)in 1-d-old and 21-d-old offspring broilers,and the activities of citroyl synthetase(CS)in breeder roosters and 21-d-old offspring broilers were all significantly increased.Taken together,our results suggested that dietary folate supplement leads to transgenerational alterations of lipid and carbohydrate metabolic process.Exp.2 Folate supplement affects the metabolomic profiles of livers samples from breeding cocks and offspring broilersBase on the phenotype changes induced by different concentration of folate supplement,we selected out the 1.2 mg/kg folate supplementary group and the control group to study the potential roles of dietary folate supplement in regulating lipid and carbohydrate metabolic process.Herein,we further studied the metabolomic changes of chicken livers using a gas chromatography-mass spectrometer(GC-MS)-based metabolomic study.In the present study,the metabolic changes in the liver between the chickens(breeder roosters,their 1-d-old and 21-d-old offspring broilers)from control and folate supplementary groups were separately examined and compared in this study.with the criteria of the variable importance in the projection(VIP)value of OPLS-DA model larger than 1 and P values of univariate statistical analysis lower than 0.05,a total of 16,15,and 7 significantly differential metabolites were separately identified from the breeder roosters,1-d-old offspring broilers,and 21-d-old offspring broilers between the control and folate supplementary group.Our results indicated several metabolites involved in different metabolic pathways were altered with the folate supplement.In order to systematically analyze the transgenerational roles of folate supplement,we firstly analyze the alteration of metabolites and its related metabolic pathway changes of breeder roosters and their offspring separately.For breeder roosters,among those 16 metabolites,we found that only 4 metabolites were significantly increased in folate supplementary group,including pantothenic acid,ethanolamine,xanthine,and uracil,which have been proved to be involved in the roles of folate in one carbon metabolism and related nucleic acid metabolism.However,another 12 metabolites were significantly decreased and were mostly related to the Glycolysis/ Gluconeogenesis,Pentose phosphate pathway,fatty acid metabolism,and purine metabolism.Following,the involved metabolic pathways were further mapped with identified significantly altered metabolites.As results,14 metabolic pathways involved in lipid metabolism and glycometabolism were standing out.Specifically,pentose phosphate pathway,purine metabolism,pantothenate and CoA biosynthesis,and glycolysis or gluconeogenesis pathway were significantly enriched,which indicated that folate supplement for breeder roosters could significantly influence the lipid,glucose,and nucleic acid metabolism process.When surfing to 1-d-old offspring broilers,among those significantly differential 15 metabolites,we found that pantothenic acid,histidine,lysine,and cysteamine were significantly increased in folate supplement group.And other metabolites,including lactic acid,2-phosphoglyceric acid,3-phosphoglyceric acid,dihydroxyacetone phosphate,tetradecanoic acid,and 1-linoleoylglycerol,which were mainly related to glycolysis or gluconeogenesis and lipid metabolism process,were all decreased in the folate supplement group.Further pathway analyses have identified 17 metabolic pathway,of these,three pathways including fructose and mannose metabolism,glycolysis or gluconeogenesis,and biotin metabolism were significantly enriched.As for 21-d-old offspring broilers,only 7 metabolites were significantly differential,and all these metabolites were involved in Fatty acid metabolism and Glycerolipid metabolism.further pathway analysis have also found that Linoleic acid metabolism,Purine metabolism,Starch and sucrose metabolism,and Glycerolipid metabolism were significantly enriched.To sum up,combined with the analysis of the breeder roosters,their 1-d-old and 21-d-old broilers,we can easily find that the folate could play important roles in regulating lipid metabolism,glycometabolism,and Purine metabolism processes.Importantly,we also found that the dietary folate supplement of breeder roosters could transgenerational regulate these metabolic process of their offspring broilers.Exp.3 Folate supplement induces transgenerational changes in transcriptomeTo gain insights into the differential gene expression correlated with changed metabolic process caused by dietary folate supplement,we analyzed the transcriptomic changes in livers of the breeder roosters and their 1-d-old offspring broilers.We separately compared the gene expression of breeder roosters and 1-d-old offspring broilers which from control and folate supplement groups.As results,a total of 42 and 82 differentially expressed mRNAs were identified from breeder roosters and 1-d-old offspring broilers.In order to classify the roles of these differentially expressed genes,these genes in each group were assigned to gene ontology terms.For breeder roosters,we found that the significantly changed mRNAs were mainly enriched in three main biological processes,including lipid and glucose metabolic processes,immune regulatory processes,and organ development processes.Meanwhile,for 1-d-old offspring broilers,similar results were also identified.In addition,analysis based on Kyoto Encyclopedia of Genes and Genomes database(KEGG)of these differentially expressed genes highlighted the mainly changed pathway were related in the lipid and glucose metabolism processes,which were consisted with the plasma indexes and metabolomic results.Specifically,based on the results of the identified enriched pathways,we can found that the dietary folate supplement of the breeder roosters could regulate the lipid and glucose metabolism of breeder roosters and transgenerational regulate the lipid and glucose metabolism processes of their offspring broilers by both influencing the PPAR pathways.Meanwhile,the FoxO signaling pathway,Pyruvate metabolism process,and glycolysis / gluconeogenesis process were also influenced by the dietary folate supplement of breeder roosters.Furthermore,the results of KEGG analyses have also identified several enriched pathway that were related to the regulation of organ development.Then,we screened out the differentially expressed mRNAs in sperm which induced by folate supplement.As results,compared with control group,there were 111,261,68,and 208 differentially expressed mRNAs identified in four folate supplement groups.Based on these differentially expressed mRNAs,we further performed the GO and KEGG analyses.According to our identified significantly enriched 163,229,143,and 188 GO terms,we found that the mostly enriched GO terms were related to the glucose and lipid metabolism,as well as the embryo development process.Moreover,KEGG analysis have identified that all our identified differentially expressed mRNAs were involved in the pathways of embryo development process and lipid and glucose metabolism process.Combined with the alterations of organ indexes induced by dietary folate supplement,the mRNA sequencing results further proved that paternal folate supplement could regulate the organ development of their offspring.Exp.4 Differential miRNA and lncRNA expression profiles contribute to the intergenerational inheritance of the effects of dietary folate supplement on metabolic changesTo identify the roles of miRNAs in transgenerationally regulating metabolism of offspring broilers,we focused on the miRNAs expression profiles changes in breeder roosters' livers,breeder roosters' sperm,and offspring broilers' livers.There were 31 differentially expressed mi RNAs were identified between the livers of breeder roosters from folate supplementary group and control group.Meanwhile,a total of 23 differentially expressed miRNAs were identified between the livers of 1-d-old offspring broilers from control and folate supplement groups.In order to illuminate the potential roles of these differentially expressed miRNAs,we further respectively predicted the potential targets of these differentially expressed miRNAs and respectively performed the GO and KEGG analyses for these differentially expressed miRNAs.In breeder roosters,2546 targets were predicted based on those 31 differentially expressed mi RNAs.The GO analyses have identified that those 31 miRNAs could be involved in the three main biological processes,including the immune changes,lipid and glucose metabolic changes,and the organ development process.Similarly,based on 2500 target genes of 23 differentially expressed miRNAs detected in offspring broilers,further GO analyses have also identified that 23 differentially expressed miRNAs of offspring broilers were involved in regulation of immune changes,lipid and glucose metabolic changes,and the organ development process.Furthermore,the KGGG analysis were also respectively performed.As results,several significantly enriched pathway involved in the regulation of lipid and glucose metabolism process were both detected in both breeder roosters and offspring broilers,including the fatty acid metabolism,fatty acid elongation,citrate cycle(TCA cycle),and glycolysis / gluconeogenesis pathways of breeder roosters,as well as fatty acid metabolism,steroid biosynthesis,propanoate metabolism,pyruvate metabolism,and glycolysis / gluconeogenesis pathways of offspring broilers.Of these significantly enriched pathways,the fatty acid metabolism and glycolysis / gluconeogenesis pathways were both detected in significantly enriched pathways of breeder roosters and offspring broilers,which indicated that miRNAs could take part in the hepatic lipid and glucose metabolic changes of breeder roosters and offspring broilers induced by paternal chronic dietary folate supplements.These aforementioned studies have proved that miRNAs in livers could take part in the hepatic lipid and glucose metabolism of breeder roosters and offspring broilers which response to paternal dietary folate supplement.In order to further identify whether miRNAs could implicated in intergenerational inheritance of metabolic changes induced by paternal chronic dietary folate supplements,we further analyzed the expression levels of spermatozoal mi RNAs and screened out the differentially expressed mi RNAs in sperm which induced by folate supplement.Consequently,when compared with the control group,there were 81,115,208,and 92 differentially expressed mi RNAs were respectively detected in those four folate supplementary groups and there were 4294,4715,5256,and 4389 targets were predicted based on these identified differentially expressed miRNAs.In order to analyze the potential roles of these differentially expressed mi RNAs,the GO and KEGG analysis based on these differentially mi RNAs of four compared groups were performed.As results,we found that these differentially expressed miRNAs could regulate several biological process related to glucose and lipid metabolic process.Meanwhile,KEGG analyses have proved that the differentially expressed mi RNAs of four compared groups could all be significantly enriched in several pathways related to the lipid and glucose metabolism process.Specifically,the glycolysis / gluconeogenesis,fatty acid metabolism,and pyruvate metabolism were the co-enriched pathways of these four compared groups.Meanwhile,these 3 pathways could also been detected as the enriched pathways based on the hepatic differentially expressed mRNAs and the targets of the hepatic differentially expressed miRNAs of the breeder roosters and offspring broilers,which induced by the paternal dietary folate supplement.Overall,our results indicated that differential miRNA expression profiles contribute to the intergenerational inheritance of the effects of paternal dietary folate supplement on lipid and glucose metabolic changes.Similarly,with the goal to identify the potential roles of lncRNAs in regulating development or metabolism changes in response to dietary folate supplement,we further analyze the expression profiles of hepatic and spermatozoal lncRNA.Firstly,we firstly focused on the roles of lncRNA in regulating hepatic gene expression in livers of breeder roosters and offspring broilers.In the present study,there were 64 and 21 differentially expressed lncRNAs were respectively detected in the breeder cocks and offspring broilers which induced by dietary folate supplement of breeder roosters.To investigate the possible functions of these differentially expressed lncRNAs,we respectively predicted the potential targets of lncRNAs in cis-regulatory relationships and in trans-regulatory relationships,by combining with the results of differentially expressed mRNAs.Of these 64 differentially expressed lncRNAs in breeder roosters,we found 29 differentially expressed lncRNAs could cis-regulated those 46 differentially expressed mRNAs and 64 differentially expressed lncRNAs could trans-regulate 42 differentially expressed mRNAs.Combined with the GO and KEGG analyses based on the cis-and trans-regulated targets of differentially expressed lncRNAs,the biological process and pathways related to lipid and glucose metabolism process were significantly enriched in the present study,such as glycerolipid metabolism,glycerophospholipid metabolism,pantothenate and CoA biosynthesis,glycolysis / gluconeogenesis,PPAR signaling pathway,FoxO signaling pathway,Phenylalanine metabolism,and Histidine metabolism,which indicated that folate supplement could regulated the lipid and glucose metabolism process by regulating lncRNAs expression.In 1-d-old offspring broilers,15 differentially expressed lncRNAs could cisregulated 36 differentially expressed mRNAs and 21 differentially expressed lncRNAs could trans-regulate 81 differentially expressed mRNAS.Overall,considered the trans-regulated targets and the cis-regulated targets of the differentially expressed lncRNAs in broiler chickens together,further KEGG analyses have identified that these lncRNAs could regulate target gene expression,which were involved in lipid and glucose metabolism process,by influencing PPAR signaling pathway and the pyruvate metabolism process as well,which indicated that paternal folate supplement could influence the lncRNA expression of broiler chickens,and further regulate the gene expression which involved in lipid and glucose metabolism process.We further identified the potential roles of spermatozoal lncRNAs as intermediaries in regulating the intergenerational inheritance of developmental and metabolic changes induced by paternal folate supplements.As results,a total of 462,633,527,and 558 differentially expressed lncRNAs were respectively identified in four folate supplement groups when compared with control group.Moreover,the cis-regulatory and trans-regulatory targets of these differentially expressed mRNAs were also respectively predicted.Combined the trans-regulated target mRNAs with the cis-regulated target mRNAs,we further performed the KEGG analyses to identify the potential role of our identified differentially expressed lncRNAs and found that our identified differentially expressed lncRNAs could regulate the target mRNAs which were also mainly involved in the lipid metabolism and development process.Overall,our results indicated that the differentially expressed lncRNAs in sperm could be served as intermediaries in regulating the intergenerational inheritance of developmental and metabolic changes induced by paternal folate supplements.In conclusion,this model suggests dietary folate supplement could transgenerational regulate the lipid and glucose metabolism in broiler and epigenetic transmission may involve the changes of miRNA and lncRNA expression profiles. |
PDF Full Text Request