Study On The Glycosylation Mechanism Of Buffalo Preimplantation Embryos And Transgenic Cloning Buffalos

The objective of this research is to explore the glycosylation mechanism of buffalo preimplantation embryos and factors affecting the efficiency of buffalo transgenic cloning, so as to establish an effective technique system for producing transgenic cloning buffalos. Works are summarized in following.1. Methods to detect the expression of O-GlcNAc (beta-O-linked N-glycosylation, O-GlcNAcylation) gene in buffalo embryos was set up. In immunofluorescence (IF) analysis, the appropriate concentration of the first antibody was proved to be1:200and concentration of the second antibody was proved to be1:1000, in which the protein of O-GlcNAc can be observed clearly after IF staining. In order to set up a method to detect the mRNA expression of O-GlcNAc using real-time fluorescence quantitative PCR, the CDS sequences of buffalo glutamine:fructose6-phosphate amidotransferase (GFPT), O-GlcNAc transferase (OGT) and O-GlcNAc-selective N-acetyl-β-D-glucosaminidase (OGA) genes were cloned using the primers designed according to the CDS sequence of human and bovine GFPT, OGT and OGA genes in GenBank. Then, the primers were designed and synthesized for real-time fluorescence quantitative PCR according to the cDNA sequence of three buffalo genes. The band of CDS was uniform and defined as the specific PCR product of target gene segment. The liquating temperature of QRT-PCR was uniform and liquating curve displayed a single peak, indicating that the primer is efficient and can be employed for detecting the expression of GFPT, OGT and OGA genes in the early buffalo embryos.2. The expression patterns of O-GlcNAc and expression of GFPT, OGT and OGA in the development of buffalo embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) was investigated using IF and QRT-PCR. The O-GlcNAc protein displayed a similar change pattern in the development of PA, IVF and SCNT embryos from the2-cell stage to the blastocyst stage, in which the O-GlcNAc begun to increase from the2-cell stage and arrived at a peak at the blastocyst stage. The level of O-GlcNAc in IVF embryos was significantly higher than PA and SCNT embryos (P<0.05), and level of O-GlcNAc in SCNT embryos was also significantly higher than PA embryos (P<0.05). QRT-PCR revealed that the level of GFPT mRNA in the three types of embryos increased from the2-cell stage to the4-cell stage, and then decreased till to the blastocyst stage. Among the three types of embryos, the GFPT expression level in IVF embryos was significantly higher than PA embryos and SCNT embryos (P<0.05), and SCNT embryos from the8-cell stage to the blastocyst stage was significantly higher than PA embryos (P<0.05). The expression of OGT increased gradually from the2-cell to the blastocyst stage, and arrived at the highest level in the blastocyst stage in the three type embryos. The OGT expression level in IVF embryos was significantly higher than PA embryos (P<0.05) and SCNT embryos from the8-cell stage to the morula stage (P<0.05). The OGA mRNA levels decreased from the2-cell to the4-cell stage, increased from the4-cell stage to the8-celln stage, and then decreased again. The OGA expression level in IVF embryos was significantly higher than PA embryos (P<0.05) and SCNT embryos at the8-cell stage and blastocyst stage (P<0.05).3. Effects of glucosamine (GlcN) on the in vitro development of buffalo PA, IVF and NT embryos, and expression of O-GlcNAc and its related genes were investigated. When buffalo embryos derived from PA, IVF and SCNT were cultured in the medium supplemented with different concentrations of GlcN (0mmol/L,1mmol/L,2mmol/L, and4mmol/L) during0to72h, addition of2mmol/L GlcN resulted in significantly higher blastocyst yield in comparison with other groups (PA:23.75%vs13.38%; IVF:26.16%vs14.29%; SCNT:25.52%vs12.70%, P<0.05). Therer was not significant difference in cleavage rate among the groups (P>0.05). Meanwhile, the O-GlcNAc level of embryos was higher than control group (P<0.05) with the exceptions of IVF blastocysts, eight-cell and morula SCNT embryos. In addition, the expression level of GFPT was also significantly higher than control (P<0.05) with the exceptions of IVF embryos at8-cell, morula and blastocyst stage, SCNT embryos at morula and blastocyst stage. As to the OGT expression, addition of2mmol/L GlcN to culture medium from0h to72h resulted in higher expression of OGT in the three type embryos with the exceptions of PA embryos at4-cell stage, IVF embryos at blastocyst stage and SCNT embryos at2-cell and blastocyst stage. The OGA expression of embryos cultured in the medium supplemented with2mmol/L GlcN during0-72h was lower than control groups (P<0.05) with the exceptions of IVF embryos. These results indicate that supplementation of2mmol/L GlcN into medium during culture of0-72h can improve the development of buffalo embryos, increases the expression of O-GlcNAc, OGT and GFPT, decreased the expression of OGA.4. Factors affecting the transfection effciciency of somatic cells and in vitro development of buffalo transgenic cloning embryos were investigated. Plasmide containing marker gene EGFP was transfected into the buffalo fetal fibroblast cells (BFF) by CaHPO4, liposome, electro-transfection and lentivirus respectively, and the transgenic-cells were used as donor cells for nuclear transfer. The percentage of EGFP-positive cleaved oocytes (48.00%) and EGFP-positive blastocysts (18.58%) was higher in SCNT embryos derived from donor cells by electro-transfection than lentivirus infection (0%&0%), CaHPO4transfection (0%&0%) and liposome transfection (5.60%&3.72%)(P<0.01). There was not different in cleavage rate and blastocyst yield between BFF transfected one and two times with electro-transfection (71.23%vs68.37%;24.66%vs18.37%, P>0.05). The cleavage rate, EGFP-positive cleavage rate and EGFP-positive blastocyst rate of SCNT embryos reconstructed with BFF containing pEGFP-IRES-NEO were significantly higher than pEGFP-N1group (71.43%vs57.77%;39.14%vs8.35%;17.64%vs5.20%, P<0.01). The cleavage and blastocyst rate of transgenic cloning embryos from pEGFP-IRES-NEO vector digested with double enzymes was not different from the vector digested with BamH I single enzyme (37.39%vs32.41%;9.66%vs8.30%, P>0.05). There was not significantly different in the embryonic development among embryos reconstructed with fetal fibroblasts derived from different generation (6-10generation)(P>0.05). The EGFP-positive cleavage rate and blastocyst rate of embryos derived from female BFF was significantly higher than male BFF (67.63%vs42.76%;22.54%vs10.52%; P<0.05). Five transgenic cloning swamp buffalos that express GFP gene were obtained following embryo transfer and one of them died at delivery. When buffalo fibroblast cells that had been transfected with IFN, GH and PRL were used as donor cells for production of transgenic cloning embryos, there was not significantly different in the cleavage rate, EGFP-positive cleaved eggs, blastocyst yield and EGFP-positive blastocysts among the three transgenic donor cells (66.44%vs62.50%,59.23%;54.11%vs57.35%,51.63%;17.81%vs19.12%,20.65%;16.44%vs13.60%,14.67%, P>0.05), and pregnancies were established following transfer of these embryos into recipients. In conclusions of these results,(1) electro-transfection is more suitable in preparing transgenic cloning buffalos than lentivirus, liposome and CaHPO4, and transgenic cloning buffalos can were obtained using this method;(2) buffalo transgenic cloning efficiency is related to the donor cell sex rather than transfection times, enzyme digestion method of vectors and passages of donor cells, female donor cells are superior to male donor cells;(3) buffalo transgenic cloning embryos can be produced using the plasmide containing IFN, GH or PRL genes, and can develop further after transfer into recipients.5Effects of TSA on the development of buffalo transgenic cloning embryos and EGFP expression were investigated. Results of flow cytometry analysis indicated that percentage of cells expressed EGFP trended to decrease with the increase of cell passages (7-11passages). Treatment of cells with5nmol/L,10nmol/L,25nmol/L or50nmol/L TSA resulted in higher proportion of G0/G1cells and EGFP-positive cells (P<0.05). In addition, treatment of cells with5nmol/L or10nmol/L decreased their DNA methylation and increased their histone acetylation (P<0.05). When donor cells were treated with10nmol/L TSA for24h before nuclear transfer, the cleavage rate (85.71%vs60.40%), EGFP-positive cleavage eggs (84.82%vs55.45%), blastocyst yield (49.11%vs26.73%) and EGFP-positive blastocysts (49.11%vs21.78%) were increased in comparison with the control group (P<0.05). When the transgenic cloning embryos were treated with5nmol/L,25nmol/L,50nmol/L and100nmol/L TSA for6h respectively, treatment with50nmol/L TSA resulted in higher cleavage rate (83.33%vs69.70%), EGFP-positive cleaved eggs (82.41%vs60.61%), blastocyst yield (45.37%vs29.29%) and EGFP-positive blastocysts (43.52%vs21.21%) in comparison with control group (P<0.05). When reconstructed embryos were treated with50nmol/L TSA for3h,6h,9h and12h, treatment for9h resulted in higher cleavage rate (86.17%vs74.31%), EGFP-positive cleaved eggs (82.20%vs70.64%), blastocyst yield (49.15%vs33.94%) and EGFP-positive blastocysts (47.46%vs32.11%) in comparison with control group (P<0.05). In addition, treatment of transgenic cloning embryos with50nmol/L TSA for9h increased their acetylation level of histone and HAT1expression, decreased the expression of HDAC1(P<0.05). In conclusions,(1) proportion of BFF expressed EGFP trends to decrease as their passage number increases;(2) treatment of donor cells with10nmol/L TSA can increase their proportion in G0/G1phases, expressed EGFP and H3K14acetylation of histone, reduce DNA methylation level, and then improve the development of transgenic cloning embryos reconstructed with these cells;(3) treatment of transgenic cloning embryos with50nmol/L TSA for9can increase their histone acetylation level, expression level of HAT1, reduce the expression of FIDAC1,and then improve their subsequent development.