| Buffalo is a valuable large livestock with great developmental potential which distributed in the southern of China,however,the milk production performance of local buffaloes is low and is in urgent need of improvement.Through the somatic cell cloning technology,buffalo individuals with excellent production performance can be replicated,and the a high-yield buffalo population can be createded quickly.However,the efficiency of buffalo somatic cell cloning is still lowly,which is not yet possible to apply as a conventional breeding technique.More and more studies have shown that the same type of somatic cells from different individuals have significant different cloning efficiencies,and the screening of animals with high cloning efficiency is one of the effective ways to improve the cloning efficiency.The similar phenomena were also found in the study on the somatic cloning of buffalo.If we can clarify the mechanism leading to the different cloning efficiency,it is expected to screen the buffalo individuals with high cloning efficiency and regulate the process of somatic cells reprogramming,which can significantly improve the efficiency of buffalo somatic cell cloning.Therefore,this study is undertaken to investigate the cellular ultrastructure,epigenetic modification and energy metabolism characteristics of buffalo donor cell lines with different cloning efficiency.Meanwhile,the factors and phenotype related to cloning efficiency of buffalo somatic cells will also be explored in this study.These research works will assist us to clarified the mechanism of donor individuals displaying different cloning efficiency,and provides a theoretical basis for screening the buffalo individuals with high cloning efficiency and artificially regulating the process of somatic cell reprogramming,improving buffalo somatic cell cloning efficiency.The results of all the works are summarized in the following:1.Biological characteristics and cloning efficiency of donor cell lines from different buffalo individualsFour buffalo fetal fibroblast cell lines(named BFF 1,BFF 2,BFF 3,BFF 4)were isolated and established.The proliferation rate of BFF 2 was faster,but there was no significant difference among these four cell lines(P>0.05).The apoptosis rate of cells isolated from different individuals was significantly different(P<0.05).The apoptosis rate of BFF 1 was significantly higher than that of other cell lines.However,there was no significant difference in chromosome stability among the four cell lines(P>0.05).The blastocyst developmental rate of cloned embryos with BFF 2 and BFF 3 as donors was significantly higher than that of cloned embryos derived from BFF 1 and BFF 4(P<0.05).2.Ultrastructural,epigenetic and metabolic states of donor cell lines from different buffalo individualsThe ultrastructural,epigenetic and metabolic states of the four BFFs were analysis respectively.Results showed that:(1)donor cells with high cloning efficiency(BFF 2,BFF 3)have less heterochromatin and larger nucleoli in the nucleus,fewer mitochondria in cytoplasm;whereas cells with low cloning efficiency are the opposite.(2)The expression levels of hetcrochromatin HP1a were lower in BFF 2 and BFF 3,and the expression level of heterochromatin relaxing factor Gadd45a was higher in BFF 2.(3)Compared with that of other cell lines,the modification of DNA methylation level(5-mc)and H3K9me3 were lower and the 5-hmC was higher in BFF2,but the difference was not significant(P>0.05).Conversely,The H3K9ac modification levels of BFF 2 and BFF 3 were significantly higher than those of BFF 1 and BFF 4 with low cloning efficiency.(4)BFF 2 and BFF 3 with high cloning efficiency were metabolized vigorously,and the rates of oxygen consumption and extracellular acidification rate were significantly higher than those of BFF 1 and BFF 4 with low cloning efficiency.Specifically,the glycolysis metabolism of BFF 2 was stronger than other cell lines.3.Screening and analysis of H3K9 trimethylation and acetylation modification loci in buffalo donor cells with significant differences of cloning efficiencyThe ChIP-seq technique was employed to detect the distribution of H3K9me3 and H3K9ac histone modifications in two buffalo fetal fibroblast cell lines(BFF 1,BFF 2)with different cloning efficiency,and then the different of modification loci were analyzed.It was found that BFF 1 has 358 unique H3K9me3 modification loci genes,4864 unique H3K9ac modification loci genes;BFF 2 has 13 specific H3K9me3 modification loci genes,and 137 unique H3K9ac modification loci genes.The GO cluster analysis of H3K9me3 modified differential loci showed that the specific differential genes in BFF1 were most closely related to metabolic processes,while the differential genes in BFF 2 were most closely related to mitochondrial function.The GO clustering analysis of H3K9ac modified differential loci showed that the differential genes of both samples were most closely related to the metabolic process,in which H3K9ac regulates the negatively related genes of glycolysis in BFF1,while in BFF2 samples,H3K9ac regulates glycolysis,ATP activity related genes.The results of KEGG functional enrichment analysis showed that the difference of H3K9me3 modification loci was related to the degradation of glycan;and the most significant difference of H3K9ac modification loci was a class of genes involved in the regulation of metabolic pathways.4.Correlation between glucose metabolism pathway and histone acetylation modification in buffalo donor cellsAfter cultured with glycolytic inhibitor 2-deoxyglucose(5 mM)for 48 h,the heterochromatin of buffalo donor cells increased,the nucleoli became smaller,the number of mitochondria increased and the modification level of H3K9ac was decreased.When 5 mM glucose was added to the culture medium,the H3K9ac modification level of the cells was increased.The glycolysis-related enzyme activity,expression level of related genes and concentration of intracellular glycolysis metabolites in the four donor cell lines with different cloning efficiency were detected,results showed that the GAPDH and LDH activities were significantly higher in BFF 2 than other cell lines.The expression level of glycolytic related genes was higher in cells lines with higher cloning efficiency.The concentration of lactate and acetyl-CoA were also significantly different among the four cell lines.When lactate or acetyl-CoA was added to culture medium,results showed that that 40 mM lactate could significantly improve the H3K9ac.modification level and reduce the compactness of chromosomes.Adding PS48 in cell cultured medium would increase the cell proliferation rate,promote the production of lactate,and improve the H3K9ac modificationlevel.Analysis the deacetylase activity of cells,results showed that the deacetylase activity of donor cells(BFF 2,BFF 3)with high cloning efficiency was lower than that of donor cells(BFF 1,BFF 4)with low cloning efficiency;adding PS48 or lactate can significantly inhibit the deacetylase activity of BFF 1.5.Effect of histone deacetylation inhibitor(TSA?Scriptaid)on buffalo somatic cell cloning efficiencyTreatment of donor cells with 30 nM TSA or 500 nM Scriptaid can significantly increase the the cleavage rate(79.1%,72.3%vs 55.9%,P<0.05)and blastocyst rate(30.8%,26.6%vs 18.4%,P<0.05)of cloned embryos.Further analysis found that TSA or Scriptaid resulted in the following changes in ultrastructure,proliferation,metabolism and H3K9ac modification of BFFs:(1)The heterochromatin in the nucleus was significantly reduced.The nucleolus became larger and the number of mitochondria decreased.The expression level of HP la was decreased while the expression of heterochromatin-relaxing factor Gadd45a was increased.(2)The ATP production was reduced and the glycolysis metabolism was enhanced in BFFs.(3)The intracellular lactate was increased,and the deacetylase activity was significantly inhibited.the H3K9ac modification level of the BFFs was significantly increased.In conclusions,the different cloning efficiency of somatic cells from different buffalo individuals is due to the difference of energy metabolism pathway,which leads to the different of epigenetic modification and chromatin structure.Buffalo donor cells with high cloning efficiency displayed less heterochromatin,larger nucleoli,fewer mitochondria,vigorous energy metabolism,higher H3K9ac modification level,lower DNA methylation and H3K9me3 methylation modification,fewer specific modification loci of H3K9me3 and H3K9ac.Enhancing the glycolytic metabolic pathways of cells or adding lacted,the intermediate product of glycolysis,can improve the level of H3K9ac and lossen the compactness of chromatin.TSA and Scriptaid also can improving the developmental potential of cloned embryos by promoting the glycolysis metabolism of cells and elevating the modification levels of H3K9ac. |
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