Preliminary Study On The Effect And Mechanism Of Pyruvate On The Clearance Of Maternal MRNA In Mouse Preimplantation Embryos

Energy is the most basic nutrient for the development of preimplantation embryos.The imbalance of energy will lead to developmental arrest.The main reason may be that the imbalance of energy will lead to the obstacle or even failure of the maternal-to-zygotic transition(MZT)process.The orderly phase-dependent clearance of maternal mRNA is the precondition to ensure the smooth progress of MZT.Considering that both energy imbalance and abnormal maternal mRNA clearance can lead to developmental arrest in preimplantation embryos,it is reasonable to speculate that energy imbalance may interfere with maternal mRNA clearance process,but this hypothesis has not been confirmed yet.Therefore,mouse preimplantation embryos were used as the experimental model in this study to preliminarily explore the effect of energy substrate pyruvate on maternal mRNA clearance of mouse preimplantation embryos and its mechanism.The main research contents and results are as follows:Experiment 1.Effects of pyruvate on the clearance of maternal mRNA in mouse preimplantation embryosIn order to investigate the effect of pyruvate on the clearance of maternal mRNA in mouse preimplantation embryos,KSOM medium with different concentrations of pyruvate was used for embryo culture.It was found that low concentration of pyruvate(a fifth of normal)led to cell block in 2-cells,while the mitochondrial content and ROS level of embryos decreased.The results indicated that low-level pyruvate reduced the metabolic activity of embryos and caused developmental arrest.Real-time quantitative reverse transcription PCR detection showed that low level of pyruvate inhibited the clearance of maternal transcripts such as Zar1 and Lrrc17 at the 2-/4-cell stage.Moreover,Btg4 and other transcripts which involved in maternal factor-mediated mRNA decay(M-decay)pathway was inhibited,while some transcripts involved in zygotic factor-mediated mRNA decay(Z-decay)pathways,such as Tead4,were reduced.And the results of transcriptional activation markers p S2 and p S5 showed that low-level pyruvate caused the decrease of fetal transcriptional activity,indicating that low-level pyruvate inhibited the transcriptional activity of early embryos.Our data reveal that low-level pyruvate may inhibit maternal mRNA clearance by interfering with the M-and Z-decay pathways,leading to abnormal embryonic development.Experiment 2.Effects of pyruvate on epigenetic modifications of mouse preimplantation embryosIn order to investigate whether energy mediated the clearance of maternal mRNA in preimplantation embryos through epigenetic modification,the modification levels of DNA methylation,histone methylation andRNA m~6A methylation in 2-cell stage mouse preimplantation embryos were detected by immunofluorescence staining.The results showed that low level of pyruvate could decrease the modification levels of 5m C,H3K4me2,H3K9me2 and H3K27me2and increase the modification level of m~6A in the early preimplantation embryos.Then,Real-time quantitative reverse transcription PCR was used to detect the expression levels of these epigenetic modification related genes,and it was found that low level of pyruvate led to abnormal expression of related genes,which further indicated that the epigenetic modification was interfered with.These results suggested that low level of pyruvate led to abnormal epigenetic modifications of mouse preimplantation embryos,which may be the potential mechanism of pyruvate-mediated clearance of maternal mRNA.In conclusion,our data revealed that pyruvate plays an important role in the orderly stage-dependent clearance of maternal mRNA in the development of mouse preimplantation embryos,which ensures the coordination of M-and Z-decay.It was also preliminarily elucidated that pyruvate may mediate the clearance of maternal mRNA in preimplantation embryos through epigenetic modification.Our results extended the current understanding of energy requirements for early embryo development and provided practical significance and theoretical basis for the improvement of embryo in vitro culture system.