Metabolic Reprogramming And Regulation Of Mouse Early Embryo Development

Growth and proliferation of cells dynamically change during the early embryogenesis of mammals.On one hand,transcription of m RNAs is involved in the regulation of development,and on the other hand,the epigenetic and metabolic changes also occur dynamically during development.The molecular regulation mechanism of this process is important because it is directly involved in the activation of embryonic genome and the determination of cell fate.In addition,there is a synergistic effect between metabolic and epigenetic regulation to participate in and promote the development of the early embryo.With the development of low-input cell technologies such as low-input Ch IP-seq,some studies have characterized the epigenetic changes during embryo development,but little is known about the metabolism of early embryo development.Therefore,we used a newly developed low-input cell metabolomics approach to analyze the metabolic profile of early embryo development and to analyze how some important metabolites in early embryos actively influence epigenetic remodeling during embryo development.The study of metabolic regulation and epigenetic regulation during embryonic development will provide important knowledge for our understanding of embryonic development,an important physiological process,as well as embryonic stem cells,which play an important role in regenerative medicine.In addition,research on the regulation of embryo metabolism and genes can enhance our understanding of how the nutritional conditions of embryos in vitro affect embryo development,thus improving the efficiency of key technologies inartificial assisted reproduction,such as in vitro fertilization and embryo culture.Firstly,we analyzed public databases of mouse oocytes and early embryos at different stages and built the metabolic network of these stages and identified some important transcription factors that regulate a metabolic network diagram of these stages.The results showed that the complexity of the network increased with the development of the embryo after fertilization.This provides a theoretical basis for us to use early embryo for metabolomics.Then,we collected embryos at two important stages,the 2-cell stage and the blastocyst stage for metabonomics.The 2-cell stage is the stage of activation of the zygote genome,and the blastocyst embryo contains ICM from which ES cells are derived.In addition,we also collected their 2-cell-like and embryonic stem cell counterparts in vitro.The results of metabonomics showed that the 2-cell embryos had different metabolic preferences from the blastocyst stage embryos.Through pathway enrichment analysis of metabolites with higher expression at this stage,we found that 2-cell embryos and 2-cell like embryonic stem cells tended to utilize methionine,polyamine and phosphatidylinositol metabolism and were in a more reductive state,while blastocysts and embryonic stem cells mainly utilized the mitochondrial TCA cycle and were in a more oxidized state.In addition,we also found that there is a reciprocal relationship between the alpha ketoglutaric acid(α-KG)and its antagonist L-2-hydroxyglutarate(L-2-HG)in oocytes,fertilized eggs and 2 cell embryos,namely,there is higher L-2-HG in oocytes,while in the blastocyst,there is higher TCA intermediate of α-KG,whereas L-2-HG content is extremely low.α-KG plays an important role in demethylation of embryonic stem cells,and L-2-HG,as a competitive inhibitor of α-KG-dependent dioxygenases,blocks demethylation.The changes between α-KG and L-2-HG are consistent with the need to erase histone methylation and DNA methylation early in the embryo.To verify that the reduction of L-2-HG is necessary for the development of embryos,we added L-2-HG with a similar concentration in vivo to the embryo culture system in vitro and found that the normal process of the early development of embryos was impaired,also the hatching rate of the formed blastocysts was reduced.As an antagonist of α-KG,which is a required co-factor for dioxygenase enzymes that include the Jumonji C(Jmj C)domain-containing histone demethylases(JHDMs)and Ten eleven translocation(TET)DNA methylcytosine hydroxylases,L-2-HG may affect embryonic development by impeding demethylation in early embryos.By investigating the change of epigenetic and the demethylase in mouse early embryo,we found that the H3K4me3 and H3K9me3 modifications were erased from fertilization to 4-cell stage.Immunofluorescence showed that L-2-HG mainly blocked these specific histones.In addition,through genetic knockdown of L2hgdh(a gene encoding L-2-hydroxyglutarate dehydrogenase that removes L-2-HG)in the zygote,we found that the abundance of 2-HG increase and the epigenetic changes were consistent with the addition of L-2-HG.Additionally,the knockdown of L2 hgdh and L-2-HG supplementation had accumulative effects.In conclusion,we found that metabolic characteristics are different between 2-cell stages and blastocysts.In addition,the study on the metabolite L-2-HG indicated that metabolites in embryonic development can actively participate in the regulation of epigenetic remodeling during development.Therefore,metabolism is dynamic during early embryonic development and is involved in the remodeling of metabolic,genetic,and epigenetic regulatory networks.On the one hand,this study enriched the knowledge of early embryo development,and on the other hand,it provided the theoretical basis for embryo culture in vitro.