Function Of MAT2A/MAT2B On Mouse Oocyte Maturation And Early Embryo Development

Mammalian oocyte maturation and early embryonic development are regulated by numerous signaling pathways and transcription factors.The maturation of the oocyte requires two meiotic processes and is finally blocked in the MII phase until fertilization.During meiosis,the MAPK signaling pathway is involved in the regulation of spindle assembly and normal alignment of chromosomes.Embryo pre-implantation development involves two key processes,one is the zygote genome activation,which is a critical period from maternal regulation to embryo regulation.Another important event in early embryonic development is the morula-blastocyst transformation,which involves the differentiation of inner cell masses and trophoblasts.Transcription factors and epigenetics modification factors play an important role in these two processes.Dissecing tthe role of key regulatory factors in oocyte maturation or early embryo development is the basis for revealing the complexity and systemic nature of developmental processes.MATII is a key complex for the metabolism of methionine in mammals.It is composed of the catalytic subunit ? and the regulatory subunit ?.Among them,the ? subunit is encoded by the MAT2A gene;the ? subunit is encoded by the MAT2 B gene.The complex forms S-adenosylmethionine(SAM)by catalyzing methionine and is closely related to the body's transmethyl metabolic pathway,and SAM is also the most important source of methyl groups in vivo.In this study,the expression patterns of MAT2A and MAT2 B genes in mouse oocytes and early embryo were studied by screening and functional analysis,and the function of MAT2 B gene in mouse oocyte maturation was studied.Meanwhile,the function of MAT2A gene in mouse early embryo development was also studied.The results are as follows:(1)The expression patterns of MAT2A and MAT2 B in mouse oocytes and preimplantation embryos were determined by quantitative and immunofluorescence staining experiments.The results showed that the MAT2 B gene was highly expressed in the GV phase and MII phase,and MAT2 B protein showed nuclear localization in the GV phase.The MAT2A gene showed high expression from MII stage to morula stage,and its mRNA level began to decrease in blastocyst stage.Immunofluorescence staining analysis showed that MAT2A protein showed obvious nuclear localization from 2-cell stage to blastocyst.(2)The effect of silencing MAT2 B on oocyte meiosis was studied by microinjection of interference fragments.The results showed that the maturation rate of mouse oocytes was significantly decreased after knocking down MAT2 B,and rate of MI/AI was significantly increased.Silencing MAT2 B significantly inhibited the phosphorylation of ERK1/2 and affected the normal function of the MAPK pathway.Further studies have found that MAT2 B protein promotes phosphorylation of ERK1/2 by interacting with GIT1,thereby regulating the function of downstream proteins.At the same time,knockdown of the MAT2 B gene affects the degradation of maternal mRNA of Fgf8,Cdc2 and Gdf9.And abnormal degradation of these mRNAs may also adversely affect the meiosis of oocytes.(3)The effect of silencing MAT2A on zygotic genome activation was studied by interference fragment microinjection.It was found that embryos was arrested at 2-cell stage after knocking down MAT2A.Silencing MAT2A reduced the overall transcriptional level of 2-cell embryos,increased DNA damage,and decreased expression level of zygotic genes.In addition,the arrested 2-cell embryos caused by MAT2A silencing can be rescued by microinjection of MAT2A mRNA and partially developed to the blastocyst stage.(4)The catalytic function of MAT2A protein in blastocyst development was studied by adding MAT2A protein inhibitor and methionine starvation culture.It was found that inhibition of the catalytic function of MAT2A or methionine starvation significantly reduced blastocyst rate,and blocked the early embryonic development into morula stage.Similar results was obtained by microinjecting the mutant MAT2A mRNA.This indicates that the catalytic function of MAT2A protein is crucial for the morula-blastocyst transition.Further studies have found that inhibition of the catalytic function of MAT2A protein or methionine starvation leads to a decreased H3K4me3 level in morula stage,indicating that the formation of SAM catalyzes by MAT2A is crucial for the normal establishment of epigenome in morula stage.Taken together,this study demonstrated that MAT2 B regulates meiotic maturation of mouse oocytes by regulating MAPK signaling pathway.And silencing MAT2 B caused abnormal arrest in MI/AI phase.On the other hand,the effects of MAT2A on mouse zygotic genomic activation and the effect of MAT2A catalytic function on morula-blastocyst transition were also studied.It was verified that MAT2A is involved in the regulation of zygotic genome activation as a transcription factor,and MAT2A is involved in the establishment of morula epigenome as an enzyme.