| At present,there is little or no available treatment for mitochondrial disease.Several assisted reproductive techniques have been proposed to help affected women prevent transmission of mutated mt DNA to their children,including oocyte donation,preimplantation genetic diagnose and mitochondrial replacement therapy.Since women couldn't have genetic relationship with their children through oocyte donation,and it is difficult to provide accurate genetic counseling based on preimplantation genetic diagnose.Now most studies focused on using mitochondrial replacement to prevent mutated mt DNA inheritance.The ultimate goal is to eliminate the transmission of mutated mt DNA by replacing defective cytoplasm with healthy cytoplasm.Thus,the determinate factor for mitochondrial replacement techniques is mutated mt DNA carryover rate.The lowest mutated mt DNA carryover rate reported was 2%.However,up to 100% mutated mt DNA was found in stem cell derived from a blastocyst with 2% mt DNA carryover.Mitochondrial autophagy is the main way for cell to eliminate mt DNA.Thus,we asked is it possible to reduce mt DNA carryover by using mitophagy? The first and most important thing to answer this question is to investigate whether there is mitophagy happen and by which pathway in implantation embryo development.Therefore,the aim of our study was to investigate mitophagy in pre-implantation embryo development.Aim1: investigation of mitophagy in preimplantation embryo development.Aim2: investigation of mitophagy pathway in preimplantation embryo development.Part 1 Investigation of mitophagy in preimplantation embryo developmentAim Mito QC-FIS1 is a PH sensitive probe,which includes m Cherry and EGFP tagged with mitochondria outer membrane protein FIS1.It is yellow(collocalization of red and green signal)in the alkaline condition.While in the acid condition,the EGFP will quench and m Cherry is stable.The PH of healthy mitochondria and lysosome are alkaline and acid,respectively.Combining the PH sensitive characteristic of mito QCFIS1 probe and the difference of PH condition between healthy mitochondria and lysosome.Studies reported mito QC-FIS1 is an effective way to observe mitophagy.Namely yellow signal represents no mitophagy and single m Cherry signal represents mitophagy.In this part,the mito QC-FIS1 probe was used to investigate mitophagy in preimplantation embryo.Method In this part,mitophagy was investigated including two parts: in vitro and in vivo.For in vitro experiment,mainly procedures are the followings: 1.Harvest PN embryos from Black6 mice.2.Two different probes were injected into PN stage embyros separately,including mito QC-FIS1(targeting mitochondria outer membrane protein FIS1)and matrixmito QC(targeting mitochondria matrix).3.Embryos were cultured in the incubator and imaged using confocal microscope.4.Single red signal observed in procedure 3 was doublechecked using Lamp1(lysosome marker)IF stainning.5.Mitophagy observed in procedure 3 was also checked using mt DNA quantification.For in vivo experiment,mitophagy reporter mouse(mito QC mouse),afforded by our coorparation lab,was used to verify mitophagy observed in in-vitro experiment.Mainly procedures are the followings: 1.Harvest the embryos at the same stage of mitophagy observed in in-vitro experiment from mito QC mouse.Directly imaged using confocal.2.The single red signal observed in procedure 2 was stained with Lamp1-far red to double check mitophagy.3.Mt DNA copy number quantification was also used to verify mitophagy observed in procedure 2.Results For in vitro experiment: 1.Mitophagy was observed in eight cell /morula and blastocyst stages by using the two models(mito QC-FIS1 and matrix-mito QC).2.By comparing the accuracy of these two models using negative control(nonmitochondria targeting mito QC probe),matrix-mito QC model showed more accurate in representing mitophagy.3.The Lamp1 IF staining results showed the collocalization of single red signal and Lamp1 far red.4.By comparing mt DNA copy number between 8cell and blastocyst stage embryos,singnificant reduce of mt DNA copy number was observed in blastocyst stage embryos.For in vivo experiment: 1.Mitophagy was observed in 8cell/morula and blastocyst stage embryos of mito QC mouse.2.The Lamp1 IF staining results showed the collocalization of single red signal and Lamp1 far red.3.By comparing mt DNA copy number between 8cell and blastocyst stage embryos,singnificant reduce of mt DNA copy number was observed in blastocyst stage embryos.Conclusion Both in vitro and in vivo experiments observed mitophagy in 8cell/morula and blastocyst stage embryos.Which were also supported by both immunofluorescence test and mt DNA copy number quantification.Part 2 Candidate mechanism of mitophagy in preimplantation embryoAim The widely reported mitophagy pathyways were mainly included the followings: PINK1-Parkin,NIX/BNIP3 and FUNDC1.The aim of our part II was to investigate candidate mechanism of mitophagy in preimplantation embryoMethods 1.We summarized all reported mitophagy related main proteins,combining with RNA-seq data of 8cell to blastocyst stage published by Petropoulos.Prodicting the expression of mitophagy related proteins in pre-implantation embryo development.2.Based on the results of method 1,the expression of NIX/BNIP3 pathway related proteins in 8cell/morula/blastocyst were higher,which is in accordance with the stages of mitophagy observed in part one.NIX and BNIP3 IF staining were used to show their expression in preimplantation embryo development.3.The mechanism of NIX/BNIP3 regulated mitophagy was reported by phosphorylation.Wild type NIX m RNA+ matrix-mito QC probe,phosphomimetric NIX m RNA+matrix-mito QC probe,wild tyoe BNIP3 m RNA and phosphomimetric BNIP3 m RNA were made using in-fusion method.4.Messages made in procedure 3 were injected into PN stage including experiment group1: wild type NIX m RNA+ matrix-mito QC probe,group 2: phosphomimetric NIX m RNA+matrix-mito QC probe,group 3: wild type BNIP3 +matrix mito QC probe,group 4:phosphomimetric BNIP3 m RNA +matrix-mito QC probe.The matrix-mito QC probe injected embryos were used as control.Mitophagy was compared between each experiment and control groups.5.Mt DNA copy number quantification was also compared between each experiment and control groups.6.NIX si RNA was also made to knowdown the expression of NIX.NIX IF was used to test the efficient of NIX si RNA.7.NIX si RNA+matrix-mito QC m RNA was injected to check whether there is any change of mitophagy.Results 1.We summarized all reported mitophagy mechanisms and found three pathways: NIX/BNIP3 pathway/FUNDC1 pathway/PINK-parkin pathway.Based on the RNA-seq data,the expression of NIX/BNIP3 and FUNDC1 pathway proteins were found in accordance with the stages of mitophagy occurred.In this chapter,we mainly focused on NIX /BNIP3 pathway.(FUNDC1 pathway was investigated by the other people in our lab)2.The IF results showed BNIP3 located in nuclear from 2cell stage to 8cell stage and began to transfer to cytoplasm from morula stage.The staining with TOM20(mitochondria marker)showed BNIP3 transferred from nuclear to cytoplasm and colocalize with TOM20.The NIX and TOM20 staining results showed NIX located in cytoplasm and the colocalization of TOM20 and NIX signals increased from morula stage.3.Compared with control group,Zeiss airyscan confocal showed more mitophagy happened in wild type NIX m RNA and phosphomimetic NIX m RNA injected embryos,especially in phosphomimetric NIX m RNA injected group.No obvious increasing of mitophagy observed in phosphomimetic m RNA injected group.4.Compared with control group,mt DNA copy number showed a significant reduce in wild type NIX m RNA and phosphomimetic NIX m RNA injected embryos,espercially in phosphomimetric NIX m RNA injected group.5.Compared with control group,no significant difference was observed in phosphomimetic BNIP3 +matrix-mito QC injected group.6.NIX IF staining showed there is no staining for NIX si RNA injected embryo.7.Compared with NIX si RNA uninjected control,there was less mitophagy in NIX si RNA injected embryos,especially for blastocysts.Conclusion Both wild type NIX and phosphomimetric NIX participate in mitophagy pathway in preimplantation embryo.And phosphorimetric NIX can induce more mitophagy... |
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