Study Of Epigenetic Modifications And Reprogramming Factors During Early Development Of Bovine Embryos

Since the birth of ‘Dolly’, somatic cell nuclear transfer (SCNT) technique rapidlyprogressed, many other animal species have been successfully cloned. However, the lowdevelopmental rate, low pregnant rate, high abortion rate of SCNT embryos and theabnormalities after birth resulted in a low efficiency of SCNT, which is a barrier to thewidespread use of this technology. Recently, the low cloning efficiency is mostly attributed tothe incomplete nuclear reprogramming of donor cells in oocyte. In order to further study theepigenetic reprogramming mechanism, and improve the efficiency of SCNT, in this study,changes of DNA methylation (5-mec), histone acetylation (H3K9ac, H4K8ac), and histonemethylation (H3K9m2, H3K9m3) during bovine oocytes in vitro maturation, IVF and SCNTembryos in vitro development were detected and analyzed systematically, and the epigeneticmodifications in bovine IVF and SCNT embryos were compared; The effect of two histonedeacetylase inhibitors, TSA and Scriptaid, on the development and epigenetic reprogrammingof bovine SCNT embryos were investigated and compared; A cell-free system in bovine MIIoocytes was established, and was used to screen the epigenetic reprogramming factors.Following results have been obtained:1. Immunofluorescence was used to detect the changes of DNA methylation (5-mec),histone acetylation (H3K9ac, H4K8ac), and histone methylation (H3K9m2, H3K9m3) duringbovine oocytes in vitro maturation, and IVF and SCNT embryos in vitro development. Theresults showed that, during bovine oocytes meiosis, the fluorescence signals of H3K9ac,H4K8ac and H3K9m2gradually disappeared from GV to MII stage, the signals of DNAmethylation and H3K9m3persistently expressed; During in vitro development of bovine IVFembryos, the fluorescence signals of DNA methylation, H3K9ac and H3K9m3graduallydecreased or disappeared from the2-cell to the8-cell stage, after zygotic gene activation (the8-cell stage), the signals gradually increased, the signal of H4K8ac highly expressed during invitro development of bovine IVF embryos, lowest at the4-cell stage, and H3K9m2signalgradually increased from the2-cell to the morula stage; During in vitro development ofbovine SCNT embryos, the fluorescence signal of DNA methylation decreased from the2-cellto the4-cell stage, and the signals gradually increased after the4-cell stage, the expression of H3K9ac stayed at a low level during the2-cell to the8-cell stage, and increased at the morulaand the blastocyst stages, the signal of H4K8ac highly expressed during in vitro developmentof bovine SCNT embryos, lowest at the4-cell stage, H3K9m2signal gradually increased fromthe2-cell to the blastocyst stage, the signal of H3K9m3gradually decreased from the2-cell tothe8-cell stage, After zygotic gene activation (8-cell stage), the signals gradually increased.These results suggested that there were general epigenetic reprogramming during bovineoocytes meiosis, and IVF and SCNT embryos in vitro development.2. The epigenetic modifications during in vitro development of bovine IVF and SCNTembryos were compared. The results showed that, DNA methylation and histone H3K9dimethylation in SCNT embryos were higher than in IVF embryos, and DNA methylationsignificant at the2-cell and the8-cell stages, and H3K9m2significant at the8-cell, the morulaand the blastocyst stages; The signal of H3K9ac in SCNT embryos was significant lower thanin IVF embryos at all the stages detected; H4K8ac signal in SCNT embryos was significantlower than in IVF embryos at the8-cell and the morula stages; H3K9m3signal in SCNTembryos was significant higher than in IVF embryos at the2-cell and the8-cell stages, butsignificant lower at the morula and the blastocyst stages. These results indicated thatabnormal epigenetic modifications existed in bovine SCNT embryos, extremely highexpression of DNA methylation, extremely low expression of histone acetylation, andaberrant expression of histone methylation, whch were induced by incomplete nuclearreprogramming, might be the reason for the abnormal development of SCNT embryos.3. The effect of TSA and Scriptaid, two different histone deacetylase inhibitors(HDACi), on in vitro development of bovine SCNT embryos were evaluated and compared.The results showed that, treatment with TSA or Scriptaid had no significant impact on themorphology of donor cell, but apoptosis rate was significantly improved, cell cycle andepigenetic modifications changed; Treatment of donor cells with TSA or Scriptaid had nosignificant impact on in vitro developmental capacity of SCNT embryos, but treatment ofreconstructed embryos with50nM TSA or500nM Scriptaid for14h could significantlyimprove in vitro developmental capacity of SCNT embryos. These results suggested thattreatment of reconstructed embryos with HDACi was more effective than treatment of donorcells with HDACi in improving in vitro developmental capacity of bovine SCNT embryos.After treatment of reconstructed embryos with TSA or Scriptaid, the signal of H3K9ac inSCNT embryos significantly increased, and the signal of DNA methylation and H3K9m2inSCNT embryos decreased, approaching the levels of IVF counterparts. These results indicatedthat HDACi could repair the abnormal epigenetic modifications of SCNT embryos andimprove the nuclear reprogramming. Treatment with TSA in high concentration significantly decreased in vitro developmental capacity of bovine SCNT embryos, suggesting that TSA hasrelatively higher cellular toxicity.4. A cell-free system from bovine MII oocytes was established. This system couldminic reprogramming in vitro and was superior for epigenetic reprogramming mechanismstudying, since a large number of cells could be induced at the same time.5. Oocytes cell-free system, two-dimensional electrophoresis and mass spectrometrytechnolocy were used to screen the candidates of nuclear reprogramming factor. Aftertreatment somatic cells with oocyte extracts,21up-regulated protein spots and15down-regulated protein spots were identified.9spots, which up-regulated in all replicates,were chosed to mass spectral analysis, and8bovine protein were identified, PRDX5，PSMB1，DDX43，GSTM3，ECI1，PAICS，EEF1G and ACTB. Those proteins involve in avariety of cellular functions, such as cellular redox, protein synthesis, protein metabolism, Gprotein-coupled receptor signaling pathway, cytoskeleton, cell proliferation and cell cycleregulation.