| Somatic cell nuclear transfer (SCNT) is a reproductive technology which has good prospects in improving and breeding work of livestock, and it becomes the hot research in biotechnology field. However, the efficiency of SCNT is still low, and there are a lot of problems which impede this technology applied in animal production, including the developmental abnormality of cloning embryos, perinatal and neonatal loss, placental abnormality or large weight children. In the past decades, the researches have revealed that the abnomal epigenetic modification is the main reason of the low efficiency in cloning embryos. Histone acetylation and DNA methylation are two key factors of epigenetic modification. Therefore, the aim of the present study is to systematically investigate that the effects of histone deacetyltransferease inhibitor Scriptaid on the developmental potential, the epigenetic modification and the embryonic gene activation of buffalo SCNT embryos, moreover, the efficiency of buffalo transgenic cloning after Scriptaid treatment is examined, in order to find an effective way for improving the efficiency of nuclear transfer and transgenic cloning in buffalo. All works are summarized in following.1. The distribution of nuclear phase of buffalo oocytes was decided by acetic orcein staining at the different culture time, and then the effect of Scriptaid on the changes of histone H3K18 acetylation and the levels of histone acetylation-related genes was detected by immunohistochemical technique and quantitative PCR (Q-PCR), respectively, the effect of Scriptaid on histone acetylation modification of oocytes was also investigated and the method of detect the expression of histone acetylation-related proteins and genes was established. The results showed:there was about 91.8% of the oocytes derived from ovarian antral follicles at the GV stage, and 83.3% of the oocytes occurred GVBD after in vitro mature culturing for 9 h; then, with the increase of mature time, the rate of MⅠ stage oocytes reached to 72.5% in the mature culturing for 15 h, and about 59.6% of oocytes entered into MⅡ stage in culturing for 22 h. Moreover, the levels of histone H3K18 acetylation of oocytes started to increase from GV to GVBD stage, but it suddenly disappeared at the MⅠ stage, and restarted to increase at the MⅡ stage; meanwhile, the fluorescent signal of histone acetylation was also detected at the first polar body. However, when buffalo oocytes were treated with 500 nmol/L Scriptaid for 24 h during mature culturing, not only the level of histone H3K18 acetylation of oocytes at the MⅠ stage was significantly improved compared with that of the non-treated group (P<0.05), the levels of histone H3K18 acetylation started to increase from GV to GVBD stage, decreased at the M I stage, and restarted to increase at the MⅡ stage. The expression levels of CBP, p300, and HAT1 gene of oocytes at various stages (GVBD, MⅠ, MⅡ) showed a increasing trend, and the expression patterns of HDAC1 and HDAC2 genes presented the wave line trend of increasing firstly and then decreasing; after 500 nmol/L Scriptaid treatment on buffalo oocytes for 24 h, the expression levels of CBP, p300 and HAT1 genes at the different stages were remarkedly improved (P<0.05), and showed a increasing trend; the expression level of HDAC2 gene was significantly decreased (P<0.05), and showed a increasing firstly and then decreasing trend; the expression level of HDAC1 gene at the various stage presented a trend of a decreasing from GV to MⅠ stage and then increasing at the MⅡ stage.2. Effects of Scriptaid on the developmental potential and the changes of epigenetic modification of buffalo SCNT embryos were investigated. Buffalo SCNT embryos were respectively treated with 0,250,500, or 750 nmol/L Scriptaid for 12 h, the development rate of blastocysts in 500 nmol/L Scriptaid-treated group was significantly higher than that of the non-treated group (19.2% vs 10.3%, P<0.05). While buffalo SCNT embryos were respectively treated with 500 nmol/L Scriptaid for different times (0 h,6 h,12 h, 18 h,24 h,30 h and 36h), the development rate of blastocysts in the embryos treated for 24 h group was remarkedly higher than those of the embryos treated for 6 h,12 h group and the non-treated group (28.2% vs 15.5%,17.4%,13.6%, P<0.05). Secondly, the histone H3K18 acetylation and global DNA methylation modification after SCNT embryos treated with Scriptaid were studied by immunohistochemical technology, the results showed the levels of histone H3K18 acetylation of SCNT embyos at various stages (2-cell,8-cell, Morula and Blastocyst) were significantly lower than those of the in vitro fertilization (IVF) group (P<0.05), and the levels of global DNA methylation in the SCNT group were significantly higher than those of IVF group and parthenogensis (PA) group (P<0.05). However, when SCNT embryos treated with 500nmol/L Scriptaid for 24h, the fluorescent intensity of histone H3K18 acetylation at different stages was remarkably increased compared with those of control group (P<0.05), and except 8-cell stage, the levels of acetylation at the other three stages were similar to those of IVF group (P>0.05). Conversely, the immunofluorescent signals for global DNA methylation was significantly decreased in the Scriptaid-treated group (P<0.05), and presented a pattern similar to that of the IVF embryos at the blastocyst stage (P>0.05). Additionally, the expression levels of histone acetylation (CBP, p300, HAT1, HDAC1 and HDAC2), DNA methylation (Dnmt1, Dnmt3a and Dnmt3b) and embryonic development (Oct-4, Nanog, Cx43 and Cdx2) related genes of embryos were also examined by Q-PCR, and found that after Scriptaid treatment on reconstructed embyos, the expression levels of CBP, HDAC1 and HDAC2 genes at 8-cell stage were similar to those of IVF group (P>0.05), the expression of Dnmt1, Dnmt3a and Dnmt3b genes was significantly decreased compared with the non-treated group (P<0.05), and the expression of Dnmtl gene was similar to that of IVF group (P>0.05). Futhermore, and no difference was detected in the expression level of Dnmtl gene between Scriptaid-treated group and IVF group (P>0.05); at the morula stage, the expression levels of Dnmtl, Dnmt3a and Dnmt3b genes were remarkedly improved after SCNT embryos treated with Scriptaid (P<0.05), and the expression of Dnmtl and CBP genes was similar to that of IVF group (P>0.05); at the blastocyst stage, the expression of Dnmt3a gene was significantly reduced in the Scriptaid-treated group (P<0.05), and the expression of Dnmt3a and HAT1 genes reached to those of IVF group (P>0.05). Interestingly, the expressions of CBP, p300, HAT1 and HDAC1 genes had a high level at the different stages of PA embryos. Meanwhile, the expression of Oct-4 gene of Scriptaid-treated SCNT embryos at the different stages was significantly increased, furthermore, the expression of Nanog gene at 8-cell and blastocyst stage, Cx43 gene at morula stage, as well as Cdx2 gene at blastocyst stage was also significantly improved than those of non-treated group (P<0.05), which reached to the expression level of IVF group.3. Effects of Scriptaid on zygote genes activation (ZGA) of buffalo SCNT embryos were investigated. It was found that in vitro fertilization embryos of buffalo were arrested in the 8-16 cell stage by 25μg/ml a-amanition. The change of embryonic nucleoli at various stages (4-cell,8-cell and Morula) was observed by Transmission electron microscopy, the results showed:In IVF embryos, the nucleolus precursor body (NPB) appeared at the 4-cell stage, the network structure of low electron density appeared around the NPB at the 8-cell stage, and NPB disappeared to form the network of transcription function at the morula stage. However, the network structure around the NPB was not found at 8-cell stage of buffalo SCNT embryos in non-treated group, while SCNT embryos treated with 500 nmol/L Scriptaid for 24h, the network structure around NPB appeared in the nuclus of 8-cell stage, and started to transcript. Moreover, the expression patterns of RNA Pol Ⅱ and fibrillarin protein at different stages (2-cell,4-cell,8-cell and Morula) of embryos were examined by immunohistochemical technique, and found that the fluorescent signal of RNA Pol Ⅱ and fibrillarin protein was not detected at the 2-cell and 4-cell stage of IVF embyos, and started to appear at the 8-cell stage. However, the fluorescent signal of the two proteins was not detected as far as morula stage in the non-treated SCNT embryos, but the weak fluorescent signal of RNA Pol Ⅱ and fibrillarin protein presented from 8-cell stage in the SCNT embryos treated with Scriptaid. Finally, the expression change of marker genes (eIF-3a and TRC) of ZGA was analysed by Q-PCR, the results showed:the expression patterns of eIF-3a and TRC genes in IVF embryos firstly presented an increasing trend from 2-cell to 8-cell stage, and drastically reduced from 8-cell to morula stage. While the expression of eIF-3a gene in the SCNT embryos showed a decreasing trend, but in the Scriptaid-treated SCNT embryos, except morula stage, the expression level of eIF-3a gene at the other three stages (2-cell,4-cell,8-cell) was significantly higher than those of the IVF group and non-treated SCNT group (P<0.05), and the highest level was present at the 8-cell stage. However, the expression level of TRC gene of SCNT embryos in the Scriptaid-treated group and non-treated group was lower compared with that of IVF group (P<0.05).4. Effects of Scriptaid on the efficiency of buffalo transgenic cloning were investigated. When buffalo fetal fibroblasts (BFF) transfered PRL gene were respectively treated with various concentration of Scriptaid (0,250,500,750 nmol/L) for different time (0,24,48,72 h), the fluorescent intensity of GFP protein of transgenic BFF treated with 250 nmol/L or 500 nmol/L Scriptaid for 72 h was improved than those of the other groups by the fluorescence microscopy observing. The results of Q-PCR analysis also showed:the mRNA expression level of GFP gene of transgenic BFF treated with 250 nmol/L Scriptaid for 72 h was remarkably higher comparing with the other treatment groups (P<0.05). Meanwhile, the results of flow cytometry detection also found that the rates of GFP-positive cells in the 250 nmol/L Scriptaid for 72 h,500 nmol/L Scriptaid for 48 h or 72 h,750 nmol/L Scriptaid for 48 h groups were significantly higher than those of the 250 nmol/L Scriptaid for 48 h,500 nmol/L or 750 nmol/L Scriptaid for 24 h group (79.8%,80.9%,80.1%,80.8%vs 73.2%, 73.1%,68.5%, P<0.05), and no difference was detected among of the other groups (P>0.05). Furthermore, the methylation of exogenous gene EF-1A promoter region in transgenic cells was examined by the bisulfite-sequencing method, and found that the methylation level of EF-1A promoter region in transgenic cells treated with Scriptaid was significantly decreased compared with that of the non-treated group (P<0.05), and there was no significant difference among of the Scriptaid-treated groups (P>0.05). Finally, the BFF transfered PRL gene treated with 250 nmol/L Scriptaid for 72 h was used for donor cells to yield the SCNT embryos, subsequently SCNT embryos were treated with 500 nmol/L Scriptaid for 24 h, we found that the development rate of blastocysts was remarkably higher compared with the non-treated group (31.3% vs 20.4%, P<0.05), moreover, the pregnancy rate after embryos transfer was significantly improved from 11.1% to 22.9%, and one of the pregnant recipients successfully given birth to one calf. |
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