| The low cloning efficiency of somatic cell nuclear transfer (SCNT) has limited itsapplication in many areas and incomplete reprogramming of donor cell nucleus is regarded asthe primary reason for low development rate, high morbidity and mortality in SCNT animals.As an important role in reprogramming, epigenetic modifications are incompletely erased inSCNT embryos, which might result in abnormal epigenetic modifications, such as higherlevel of DNA methylation, lower level of histone acetylation and aberrant histonemethylations. Histone methylations have complicated modifications involved in three kind ofmethylations (mono-, di-and trimethylation) and different lysine sites to be modified. Duringthe development of embryos, H3K9methylationa are associated with the heterochromatinformation and transcription silence, of which H3K9dimthylation (H3K9me2) is involved ingene silence in euchromatin. Conversely, H3K4dimethylation (H3K4me2) is correlated withtranscription activation. Compare with in vitro fertilization (IVF) embryos, in previous studies,the abnormal H3K9me2and H3K4me2were observed in SCNT embryos at2-cell stage andthis tendency maintained in subsequent in vitro development, which indicates that incompleteerasion or reconstruction of H3K9me2and H3K4me2occured during the reprogrammingprocess of donor cell nucleus. Similarly, Oct4was not completely reactivated in clonedembryos. Both of H3K9me2and H3K4me2could respectively induce the repression andactivation of Oct4and, therefore, the higher level of H3K9me2and lower level of H3K4me2probably lead to the incomplete reactivation of Oct4in SCNT embryos. The results show usthe probability of enhancing the development of SCNT embryos through correcting the twoabnormal methylations. In order to correct the abnormal H3K9me2, we used BIX01294andUNC0638to treat goat embryonic fibroblasts (GEFs) respectively, and then the in vitrodevelopment of SCNT embryos derived from UNC0638-treated GEFs was studied. Similarly,2-PCPA was used to treat GEFS to indirectly upgrade the H3K4me2levels of SCNT embryos.In this study, the abnormality of H3K9me2and H3K4me2existing in SCNT early embryoswas identified and corrected. The main results were as following:1. GEFs were treated with different concentrations of BIX01294and UNC0638, respectively and the cell viabilities, cell cycle, the effect of down-regulating H3K9me2andthe mRNA expression level of pluripotent genes were tested. The results showed that bothBIX01294and UNC0638could improve the percentage of G0/G1cells, down-regulatingH3K9me2levels and significantly improve the mRNA expression of Oct4and Nanog. Then,UNC0638was preferred for subsequent donor cell treatment because of its betterdown-regulating effect, and the concentration is0.2μmol/L.2. The in vitro development of cloned embryos which were derived fromUNC0638-treated GEFs was studied. The results showed that, compare with intracytoplasmicsperm injection (ICSI) embryos, SCNT embryos at the same stage had higher levels ofH3K9me2. However, the H3K9me2levels of SCNT embryos derived from UNC0638-treateddonor cells were down-regulated and did not differed significantly with ICSI embryos at thesame stage. In other words, UNC0638had the ability of indirectly correcting the abnormalmodification of H3K9me2in SCNT embryos. Though there is no enhancement about in vitrodevelopment rate observed, UNC0638was able to enhance the mRNA expression of Oct4andNanog in cloned blastocysts, which indicated that the quality of SCNT blastocysts wasimproved.3. GEFs treated with different concentrations of2-PCPA was observed. The resultsshowed that2-PCPA was able to induce cell cycle arrest, and significantly upgrade the levelof H3K4me2and improve the mRNA expression of Oct4, Sox2and Nanog when theconcentration over than2μmol/L of2-PCPA was used.4. The in vitro development of SCNT embryos derived from2-PCPA-treated GEFs wasstudied and the results showd that2-PCPA was able to enhance the in vitro development rateof SCNT blastocysts significantly, and improve the mRNA expression of Oct4, Sox2andNanog. And most importantly, it could indirectly correct the lower level of H3K4me2. Theseresults showed that2-PCPA was able to improve the quality of SCNT blastocysts. |
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