Metabolic Dynamics In Mouse Preimplantation Embryos And Functional Analysis Of NAD~+

Mammalian preimplantation embryonic development encompasses the developmental stages from fertilization to implantation,experiencing several key events such as zygotic genome activation(ZGA).The ZGA of mouse preimplantation embryos mainly present with t wo transcription bursts,the minor ZGA and major ZGA.Extensive and increasing studies focus on the major ZGA,while the regulatory mechanism of the minor ZGA still remains elusive.The in-depth analysis of the early embryo developmental mechanism plays a crucial role in promoting the progress of regenerative medicine and assisted reproduction technology.In recent years,high-throughput sequencing technologies have revolutionized the understanding of embryonic development concerning transcriptomes,proteomes,and epigenomics by enabling large scale whole genome sequencing.However,metabolome of mammalian embryonic development is still limited,which demand intensive research efforts in the future.In addition,cellular metabolism underlies chromatin state and genome regulation.And metabolic intermediates often involved in epigenetic remodeling.Epigenetic modification is essential for gene expression,genomic imprinting,and cell differentiation.Although researchers have conducted numerous preliminary studies on the dynamics of epigenetic modification in early embryos,for instance,acetylation and methylation.Little is known about the interaction mechanism between metabolome and epigenome in mouse preimplantation embryos.By ultra-high-performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOFMS),we systematically monitored metabolic dynamics of mouse preimplantation embryos(about 60,000 embryos).In total,246 metabolites were quantified,which were also validated by carbon targeted metabolomic analysis.The metabolomic data showed that mouse preimplantation embryo mainly consumed maternal metabolites precede ZGA.Upon ZGA occurs,the level of metabolites gradually elevated,especially in the morula and blastocyst stages.Then,all metabolites were mapped to their respective biochemical pathways,and we observed two distinct patterns in lipid metabolism and carbon metabolism.Levels of maternal metabolites were dramatically declined when ZGA occurs.In contrast,the majority of embryonic metabolites elevated in later developmental stages.The levels of nucleic acid metabolites significantly increased at the 4-cell stage,while amino acid metabolites showing stage-specific enrichment.Combined with transcriptome and proteome data,we determined that glycolysis pathway and glutamine metabolism pathway were mainly activated at the blastocyst stage,and the citric acid(TCA)cycle and methionine cycle pathway activity were significantly enhanced at the 8-cell stage.By performed WGCNA and correlation analysis,NAD~+was identified to be the key metabolite of mouse 2-cell embryo,and chemical detection result suggested that NAD~+was largely consumed at the 2-cell stage.The analysis of NAD~+synthesis pathway showed that NAD~+was mainly derived from the salvage pathway in mouse embryos.Inhibition of the NAD~+synthesis salvage pathway(FK866-treated)led to developmental arrest at 4-to 8-cell stage,and NMN supplementation could fully rescue the embryonic developmental arrest caused by FK866.In addition,the interconversion of pyruvate and lactate also contribute to the regulation of NAD~+,of which Ldhb may play an important role.The tryptophan de novo synthesis pathway and malate-aspartate shuttle have a limited capacity to regulate NAD~+level.By switching the medium between control and FK866-treated at different time points,the critical period for the NAD~+requirement is between early 2-cell and 4-cell stage.Then,RNA-seq of FK866-treated embryos were conducted,and differential gene expression and GSEA analysis showed that minor ZGA gene excessive expression at late 2-and 4-cell stage.Q-PCR and immunofluorescence staining confirmed the overexpression of the pioneer transcription factor Dux in the late 2-cell stage.Addition of NMN could rescue the overexpression of minor ZGA genes in FK866-treated embryos.NAD~+is a crucial cofactor for deacetylase.We found that H3K9ac maintained relatively stable state during mouse preimplantation embryonic development,while H3K27ac showed a highly dynamics.H3K27ac was established in the zygote PN1 stage,which was rapidly lost from the middle to late 2-cell stages,while increased progressively after the 4-cell stage.The CUT&Tag data showed that zygotic H3K27ac was widely established on a genome-wide scale,mainly distributed in promoters and intergenic regions,and was massively erased at the late 2-cell stage.Immunofluorescence staining and CUT&Tag data showed that H3K27ac deacetylation was blocked in FK866-treated late-stage 2-and 4-cell embryos.Importantly,74.7%of the minor ZGA genes promoter regions were enriched H3K27ac modification.Minor ZGA gene H3K27ac modification remained high at the zygote and early 2-cell stage,while significantly decreased at the late 2-cell stage,accompanied by reduced chromatin accessibility.In contrast,the levels of the major ZGA genes promoter H3K27ac and accessibility reta ined low at the zygote and early 2-cell stage,and significantly increased at the late 2-cell stage.Levels of H3K27ac was highly consistent with the expression of minor ZGA genes,suggesting a regulatory role of H3K27ac for minor ZGA.Genomic profiles for Pol II confirmed the link between H3K27ac and minor ZGA gene expression.For example,overexpression of Dux in FK866-treated embryos was also accompanied by the failure of H3K27ac deacetylation.TSA is a well-known inhibitor of deacetylase,and TSA-treated embryos are mainly arrested at the 4-cell stage.We validated the role of H3K27ac deacetylation in precise temporal regulation of minor ZGA by immunofluorescence staining,CUT&Tag and transcriptome data of TSA-treated embryos.Furthermore,we screened gene candidates which were responsible for H3K27ac deacetylation and found that Sirt1 was significantly upregulated at the middle 2-cell stage.We specifically depleted Sirt1 using RNAi and trim-away,and found that Sirt1-depleted embryos showed the compromised developmental potency,accompanied by the failure of H3K27ac deacetylation and overexpression of minor ZGA genes.These results revealed that Sirt1 was involved in the erasure of H3K27ac and precise temporal regulation of minor ZGA genes at the 2-cell stage.Finally,we investigate whether a similar mechanism might operate in human embryos.Human preimplantation embryo ZGA mainly occurred at the 8-cell stage.Compared to zygotes and 2-cell stage,the H3K27ac level of human 8-cell was significantly reduced.And,the SIRT1protein coding domain is highly conserved between humans and mouse.The expression level of SIRT1 was also significantly upregulated at the 8-cell stage.The developmental rate of human embryo was significantly increased when cultured i n G1 medium that contains NMN.Importantly,NMN-treated human 8-cell embryos exhibited reduced H3K27ac levels,suggesting that NMN may be beneficial for human preimplantation embryo development.In summary,we collected a large number of embryos to charact erize the global metabolic dynamics during mouse preimplantation embryo development,and uncovered that H3K27ac deacetylation was mediated by the NAD~+-Sirt1 coordination to regulate expression of minor ZGA precisely,thus ensure the progress of embryonic development.In addition,we also verified that the above mechanism is conserved between mouse and human embryos,and demonstrated that NMN may be beneficial for human preimplantation embryonic development.Our study provides an invaluable resource and lays down a theoretical foundation for metabolomic research of mouse preimplantation embryos.Meanwhile,the interaction mechanism between the metabolome and epigenomics will opens up new dimensions and points out new direction for future research.