Study On The Effect Of ROS On The Development Block In Mouse Preimplantation Embryo

In vitro culture system influences the development potential of mammalian preimplantation embryos.In the current culture systems,which simulating the hypoxic condition in vivo of uterus and fallopian tubes,the reactive oxygen species(ROS)is considered to be a major factor affecting embryo development.Zygotic genome activated(ZGA)is critical for normal embryonic development,delay or failure of ZGA usually caused embryos development block.To study the effect of ROS on the development block in mouse preimplantation embryo,Firstly,embryos from different mouse strains were cultured in the different media,the expression of ZGA related genes,the concentration of ROS in embryo cytoplasm,and the ROS related genes were detected.Secondly,the in vitro fertilized eggs from the Kunming mouse(Mus musculus)were treated by 2,2'-Azobis(2-methylpropionamidine)dihydrochloride(AAPH),and then the mitochondrial activity were evaluated,including the mitochondrial membrane potential,the mitochondrial ROS(mtROS)and the distribution of mitochondrial,the mitochondrial DNA(mtDNA)copy number were examined;In addition,the expression of ROS related genes(Grm2?Drd2?Polg2?TFAM and the DNA methylation stutus in the regulatory region of those genes were detected.1?The relevant research between ZGA and ROS of different strains of mouse embryos in different cultureIn this study,the expression of ZGA(Zscan4?MuERV-L?Eif-1a?Hsp70.1)and ROS(Nox1?Gpx4?Gpx6?Prdx2)related genes in 2-cell mouse embryos were detected by quantitative real-time PCR;meanwhile,the content of ROS in embryo cytoplasm were detected by DCFH-DA.Firstly,in vitro fertilized eggs from Kunming strains and BDF1 strain mouse were cultured in M16 and KSOM culture media,and the development block rates were compared.The result showed in M16 medium,the 4-cell rate was 44.2%for KM mice embryos,and was 90.3%for BDF1.The expression of MuERV-L and Eif-1a in KM mice was obvious lower than that of BDF1 mice,and the expression of ROS related genes,Gpx6 and Prdx2 were higher in KM mouse than in BDF1 mouse.But the intracytoplasmic ROS levels in KM mouse 2-cell embryo were lower than that in BDF1.These results showed that the development block is associated with the expression of ZGA related genes,the differences in embryo intracytoplasmic ROS levels may indicated the different toleance to ROS.Then,the development of embryos from the block strain KM mouse were compared after cultured in M16 and KSOM media.The result showed that the 4-cell rate was 90.8%in KSOM medium,no 2-cell block occurred.The expression of Eif-1a?Hsp70.1 in 2-cell embryos were significantly higher in KSOM group than that in M16 group.there was differences in the expression of ROS related genes and ROS levels in the embryo cytoplasm.These results showed that the composition in culture media affect the expression of ZGA related gene,and impact on mouse embryo development block.2.The research on the influence of ROS over the development block of early mouse embryosIn vitro culture system influences the development potential of mammalian early embryos.In thecurrent culture systems,which simulating the hypoxic condition in vivo of uterus and fallopian tubes,thereactive oxygen species(ROS)is considered to be a major factor affecting embryo development.In this study,the effect of ROS on the mitochondrial function and the mitochondrial DNA(mtDNA)copy number in mice(Mus musculus)2-cell embryos were studied.The fertilized eggs from the Kunming mouse were treated by 2,2'-azobis(2-methylpropionamidine)dihydrochloride(AAPH),then the mitochondrial activity,the mitochondrial membrane potential,the mitochondrial ROS(mtROS)and the distribution of mitochondrial were observed byfluorescent detection,meanwhile,the expression of ROS-related genes,the mitochondrial transcription factor A(TFAM)and the mitochondrial DNA(mtDNA)copy number were detected by real-time PCR.The resultsshowed that mouse embryos had a higher blocking rate(66.16%)and a lower embryo damage rate(24 hmalformation rate of 9.09%,48 h mortality 10.39%)in 1.0 mmol/L treated group.ROS staining resultsshowed that embryonic cytoplasmic ROS levels were increased significantly after AAPH treatment,and themRNA expression of ROS-related genes were also increased.After treated by 1.0 mmol/L AAPH,themembrane potential,the mtDNA copy number and the level of mitochondrial ROS(mtROS)in 2-cell embryoswere significantly higher than that of control group,and the expression of TFAM gene was also increased.Theabove results showed that the oxidative stress caused by AAPH resulted in embryo development block,thefunction of mitochondrial was promoted after treatment,with increased the mtDNA copy number and theexpression of TFAM gene,which may participate in regulation of intracellular ROS dynamic balance inembryos.This study preliminarily explored the relationship between the ROS and the dynamic change ofmitochondrial function,and might provide references for illustrating the mechanism of oocyte maturation andalso optimization the culture system of mammalian embryos in vitro.The results from the bisulfite genomic sequencing(BSP)showed that after the treatment of AAPH,the DNA methylation stutus were increased in in the regulatory region of Grm2?Drd2 genes,while were decresed in Polg2?TFAM.In conclusion,embryos from different mouse strains have the different tolerance capability to ROS level,the development block is associated with the expression of ZGA related genes,mitochondria take a role in regulation of intracellular ROS affecting embryonic development.This study preliminarily explored the relationship between ROS and the dynamic change of mitochondrial function,and might provide references for optimization the culture system of mammalian embryos in vitro.