牦牛IVF、SCNT优化及早期胚胎差异表达基因筛选
发布时间：2018-09-07 16:34
【摘要】：牦牛是青藏高原及毗邻地区集役、肉、奶、皮、绒等为一体的特有畜种,是牧民生产和生活资料的主要来源,由于特殊自然环境的限制,牦牛生产性能及繁殖性能普遍较低。探索利用体外胚胎生产技术和体细胞核移植技术等现代生物技术应用于牦牛,提高优秀牦牛个体的扩繁速度,降低繁育成本,保存优良牦牛遗传资源,为牦牛的选育改良提供技术支持。本研究优化牦牛体外受精和体细胞核移植胚胎的培养体系;利用RNA-seq技术筛选牦牛体外受精囊胚和体细胞核移植囊胚差异表达基因,并探讨组蛋白甲基转移酶抑制剂通过促进核重编程提高牦牛克隆效率的方法及氟化钠对牛附植前胚胎发育和凋亡的影响。主要研究内容和结果如下:1.优化了牦牛体外受精、体细胞核移植体系(1)A级COCs体外成熟培养24 h,COCs成熟率、卵裂率和囊胚率显著高于B级和C级COCs;用Percoll液梯度离心分离精子法和BO液上浮法分离牦牛精子,用含3 mg/m L BSA和2.5 mM Theophylline的BO受精液中精子密度在1-5×106mL-1范围受精效果良好;受精卵用TCM 199+卵丘细胞或TCM199+输卵管上皮细胞的共培养体系效果显著高于G1/G2液和SOF液培养体系。(2)用于体细胞核移植的牦牛卵母细胞体外成熟培养20 h后去核,耳源成纤维细胞作为供体细胞传代次数为6代以内,血清饥饿2 d进行核移植;重构胚电融合参数为1.4-1.6 kv/cm、两次脉冲,脉冲时长20μs,脉冲间隔10μs;离子霉素联合6-DMAP进行激活4 h后使用G1/G2胚胎培养液进行培养。2.利用RNA-seq技术挖掘牦牛IVF和SCNT囊胚的差异表达基因(1)IVF-Blastocyst文库和SCNT-Blastocyst文库进行单细胞测序,分别获得52,365,530和52,365,908 raw reads,过滤后分别得到48,810,404(93.21%)和49,328,624(94.20%)clean reads;与牦牛基因组的比对率分别为74.18%和74.54%,其中Unique match比对率分别为64.88%和64.66%。clean reads与牦牛已知基因的比对率分别为52.36%和51.00%,其中Unique match的比对率分别为47.17%和45.25%。(2)通过比对,共有14,352个基因,其中12,395个基因共表达,837和1,120个基因分别特异性表达于ivf-blastocyst和scnt-blastocyst中。采用fdr≤0.001和abs(log2(y/x))≥1的标准共筛选出576个差异表达基因,其中342个基因高表达于scnt-blastocyst,234个基因低表达于ivf-blastocyst。(3)go富集显示,576个差异表达基因被富集于150个cellularcomponentgo项目、218个molecularfunctiongo项目和1042个biologicalprocessgo项目中;显著性分析,有4个cellularcomponentgo项目和2个biologicalprocessgo项目显著富集。经注释后表明差异基因主要参与调节胞外区(go:0044421和go:0005576)、脂蛋白复合物(go:0032994和go:0034358)以及炎症和创伤反应(go:0006954和go:0009611)。(4)kegg通路分析576个差异表达基因参与218条通路,主要包括代谢通路、癌症中的转录失调、类固醇激素生物合成、神经营养因子信号通路、补体与凝血级联和细胞粘附分子。经显著分析发现,其中仅戊糖、葡萄糖醛酸转换(ko00040)达到显著水平(qvalue0.05)。(5)随机选取20个差异表达基因进行荧光定量pcr验证,结果显示egr1、cpeb1、fgfr2、slc2a4基因在scnt组中的表达量显著上调,hspb1、wnt2b、loc102279723、cdca7l、polr2a、crabp1、kifc3、cdc25a、ddit3、sdc1、loc102282421基因在ivf组中的表达量显著上调,且与rna-seq数据基本一致。(6)ivf-blastocyst和scnt-blastocyst分别预测出1,435和1,499个新转录本,通过编码潜能预测发现分别有23.62%和22.28%的转录本被预测出具有编码潜能。3.组蛋白甲基化转移酶抑制剂对牦牛scnt胚胎发育的影响(1)低剂量gsk126(10μm和20μm)处理牦牛成纤维细胞形态和细胞活力未发生明显变化,高剂量(40μm和80μm)处理组细胞质中出现黑点,活力下降,g0/g1期比例增加、s期和g2/m期比例明显降低,凋亡细胞数增加并影响到细胞增殖和代谢。(2)gsk126处理牦牛成纤维细胞后核移植,发现低剂量组核重构胚囊胚的发育率、te细胞数和icm细胞数显著高于高剂量处理组;而囊胚中凋亡细胞数显著低于高剂量组。依据细胞活力、囊胚发育率、囊胚发育质量综合评价20μmgsk126处理牦牛成纤维细胞24h的作为供体细胞进行核移植效果最佳。(3)分析IVF囊胚、SCNT囊胚和20μM GSK126处理的SCNT囊胚中组蛋白甲基化水平,发现GSK126处理后能降低核移植囊胚中H3K27me2和H3K27me3组蛋白甲基化水平,而对H3K4me2组蛋白甲基化水平没有显著影响。(4)分析胚胎凋亡和发育相关基因,发现GSK126处理后囊胚中凋亡基因BCL-2mRNA表达水平下降,Caspase-9 mRNA表达水平上升,同时能上调胚胎发育基因NANOG、OCT4和GATA3基因mRNA的表达水平。4.NaF对附植前胚胎发育和凋亡的影响(1)NaF处理卵母细胞后,呈剂量依赖性显著抑制卵母细胞的成熟率、卵裂率和囊胚发育率;高剂量的NaF处理后卵母细胞内ROS含量显著增加并抑制卵母细胞内T-AOC、GSH-Px、CAT和GSH活性从而诱导卵母细胞氧化应激。(2)随Na F剂量的增加,卵母细胞中Caspase-3和Caspase-9活性显著增加;凋亡基因Bax、Caspase-3和Caspase-9的mRNA表达水平升高,抗凋亡基因Bcl-2mRNA表达水平降低;同时,卵母细胞内凋亡蛋白Bax表达水平增加,抗凋亡蛋白Bcl-2的表达水平则随之下降。(3)NaF处理卵母细胞后体外受精,囊胚总细胞数和滋养层细胞数以及内细胞团数量随NaF剂量的增加而显著减少,囊胚细胞内凋亡细胞数随之增加;胚胎发育相关基因Oct4、Nanog、Sox2和Cdx2的mRNA表达水平随着NaF剂量的增加而显著降低。
[Abstract]:Yak is a special breed of livestock in Qinghai-Tibet Plateau and its adjacent areas, which integrates meat, milk, skin and cashmere. It is the main source of herdsmen's production and livelihood. Due to the limitation of special natural environment, the production performance and reproductive performance of yak are generally low. In this study, we optimized the culture system of in vitro fertilization and somatic cell nuclear transfer embryos of yak, screened the embryos of in vitro fertilization blastocysts and somatic cell nuclear transfer blastocysts of yak by RNA-seq technology. The main contents and results were as follows: 1. Optimizing in vitro fertilization of yaks, somatic cell nuclear transfer system (1) A-class COCs maturation culture in vitro for 24 hours, C OCs maturation rate, cleavage rate and blastocyst rate were significantly higher than those of grade B and grade C COCs; yak sperm were separated by Percoll fluid gradient centrifugation and BO fluid floatation, and the fertilization effect was good when the sperm density of BO fertilized with 3 mg/mL BSA and 2.5 mM Theophylline ranged from 1 to 5 x 106 mL-1; fertilized eggs were fertilized with TCM 199 + cumulus cells or TCM 199 + fallopian tubes. The effect of co-culture system of epithelial cells was significantly higher than that of G1/G2 and SOF. (2) Yak oocytes used for somatic cell nuclear transfer matured in vitro and cultured for 20 hours, then were denucleated. Otological fibroblasts were subcultured within 6 generations as donor cells, and serum starved for 2 days. Secondary pulses lasted 20 microseconds with a pulse interval of 10 microseconds; ionomycin combined with 6-DMAP was activated for 4 hours and then cultured in G1/G2 embryo culture medium. 2. Differentially expressed genes (1) IVF-Blastocyst library and SCNT-Blastocyst library were digged by RNA-seq technique to obtain 52,365,530 and 52,365,9,respectively. 08 raw reads, 48,810,404 (93.21%) and 49,328,624 (94.20%) clean reads were obtained after filtration, and the ratio of clean reads to yak genome was 74.18% and 74.54%, respectively. The ratio of Unique match was 64.88% and 64.66% respectively. The ratio of clean reads to known yak genes was 52.36% and 51.00%, respectively. 47.17% and 45.25%. (2) A total of 14,352 genes were identified, of which 12,395 genes were co-expressed, 837 and 1,120 genes were specifically expressed in ivf-blastocyst and scnt-blastocyst respectively. A total of 576 differentially expressed genes were screened by FDR < 0.001 and ABS (log2 (y/x) > 1, of which 342 genes were highly expressed in scnt-blastocyst and 2,120 genes were highly expressed in scnt-blastocyst. 34 genes were low expressed in ivf-blastocyst. (3) go enrichment showed that 576 differentially expressed genes were enriched in 150 cellular component go projects, 218 molecular function go projects and 1 042 biologic process go projects; significant analysis showed that 4 cellular component go projects and 2 biologic process go projects were significantly enriched. These results indicated that the differentially expressed genes were involved in regulating extracellular domain (go:0044421 and go:0005576), lipoprotein complexes (go:0032994 and go:0034358) and inflammatory and traumatic reactions (go:0006954 and go:0009611). (4) KEGG pathway analysis showed that 576 differentially expressed genes were involved in 218 pathways, including metabolic pathways, transcriptional disorders in cancer, steroid hormone production. Biosynthesis, neurotrophic factor signaling pathway, complement cascade, coagulation cascade and cell adhesion molecule. significant analysis showed that only pentose and glucuronic acid conversion (ko00040) reached significant levels (qvalue 0.05). (5) 20 differentially expressed genes were randomly selected for fluorescence quantitative PCR verification, and the results showed that egr1, cpeb1, fgfr2, slc2a4 genes in SCNT The expression levels of hspb1, wnt2b, loc102279723, cdca7l, polr2a, crabp1, kifc3, cdc25a, ddit3, sdc1, loc102282421 genes in IVF group were significantly up-regulated, and were consistent with the RNA-seq data. (6) ivf-blastocyst and scnt-blastocyst predicted 1,435 and 1,499 new transcripts respectively. 23.62% and 22.28% of transcripts were predicted to have coding potential respectively. 3. Effects of histone methylation transferase inhibitors on embryonic development of yak SCNT (1) The morphology and cell viability of yak fibroblasts treated with low doses of gsk126 (10 and 20 microns) did not change significantly, and black spots appeared in cytoplasm of yak fibroblasts treated with high doses (40 and 80 microns). (2) After nuclear transplantation of yak fibroblasts treated with gsk126, it was found that the development rate of nuclear reconstituted blastocysts in low dose group, the number of te cells and the number of ICM cells in blastocysts were significantly higher than those in high dose group. According to the cell viability, blastocyst development rate and blastocyst development quality, the best effect of nuclear transplantation was obtained when the yak fibroblasts were treated with 20 micron gsk126 for 24 hours. (3) Histone methylation levels in IVF blastocysts, SCNT blastocysts and SCNT blastocysts treated with 20 micron GSK126 were analyzed. H3K27me2 and H3K27me3 histone methylation levels in nuclear transfer blastocysts were decreased, but H3K4me2 histone methylation levels were not significantly affected. (4) Analysis of embryo apoptosis and development-related genes, it was found that GSK126 treatment of blastocysts apoptotic gene BCL-2 mRNA expression level decreased, Caspase-9 mRNA expression level increased, and at the same time, it could up-regulate embryo development. Effects of NaF on the development and apoptosis of preimplantation embryos (1) After treated with NaF, the maturation rate, cleavage rate and blastocyst development rate of oocytes were significantly inhibited in a dose-dependent manner; ROS content in oocytes significantly increased and T-AO was inhibited after treated with high dose of NaF. The activities of C, GSH-Px, CAT and GSH induced oxidative stress in oocytes. (2) The activities of Caspase-3 and Caspase-9 in oocytes increased significantly with the increase of Na F dosage, the mRNA expression levels of apoptotic genes Bax, Caspase-3 and Caspase-9 increased, and the expression levels of anti-apoptotic genes Bcl-2 mRNA decreased. With the increase of NaF dosage, the total number of blastocysts and trophoblast cells and the number of inner cell mass decreased significantly, and the number of apoptotic cells in blastocysts increased. The mRNA expression of embryonic development-related genes Oct4, Nanog, Sox2 and CDx2 increased. The level of NaF decreased significantly with the increase of dose.
【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823.85
，
本文编号：2228810
[Abstract]:Yak is a special breed of livestock in Qinghai-Tibet Plateau and its adjacent areas, which integrates meat, milk, skin and cashmere. It is the main source of herdsmen's production and livelihood. Due to the limitation of special natural environment, the production performance and reproductive performance of yak are generally low. In this study, we optimized the culture system of in vitro fertilization and somatic cell nuclear transfer embryos of yak, screened the embryos of in vitro fertilization blastocysts and somatic cell nuclear transfer blastocysts of yak by RNA-seq technology. The main contents and results were as follows: 1. Optimizing in vitro fertilization of yaks, somatic cell nuclear transfer system (1) A-class COCs maturation culture in vitro for 24 hours, C OCs maturation rate, cleavage rate and blastocyst rate were significantly higher than those of grade B and grade C COCs; yak sperm were separated by Percoll fluid gradient centrifugation and BO fluid floatation, and the fertilization effect was good when the sperm density of BO fertilized with 3 mg/mL BSA and 2.5 mM Theophylline ranged from 1 to 5 x 106 mL-1; fertilized eggs were fertilized with TCM 199 + cumulus cells or TCM 199 + fallopian tubes. The effect of co-culture system of epithelial cells was significantly higher than that of G1/G2 and SOF. (2) Yak oocytes used for somatic cell nuclear transfer matured in vitro and cultured for 20 hours, then were denucleated. Otological fibroblasts were subcultured within 6 generations as donor cells, and serum starved for 2 days. Secondary pulses lasted 20 microseconds with a pulse interval of 10 microseconds; ionomycin combined with 6-DMAP was activated for 4 hours and then cultured in G1/G2 embryo culture medium. 2. Differentially expressed genes (1) IVF-Blastocyst library and SCNT-Blastocyst library were digged by RNA-seq technique to obtain 52,365,530 and 52,365,9,respectively. 08 raw reads, 48,810,404 (93.21%) and 49,328,624 (94.20%) clean reads were obtained after filtration, and the ratio of clean reads to yak genome was 74.18% and 74.54%, respectively. The ratio of Unique match was 64.88% and 64.66% respectively. The ratio of clean reads to known yak genes was 52.36% and 51.00%, respectively. 47.17% and 45.25%. (2) A total of 14,352 genes were identified, of which 12,395 genes were co-expressed, 837 and 1,120 genes were specifically expressed in ivf-blastocyst and scnt-blastocyst respectively. A total of 576 differentially expressed genes were screened by FDR < 0.001 and ABS (log2 (y/x) > 1, of which 342 genes were highly expressed in scnt-blastocyst and 2,120 genes were highly expressed in scnt-blastocyst. 34 genes were low expressed in ivf-blastocyst. (3) go enrichment showed that 576 differentially expressed genes were enriched in 150 cellular component go projects, 218 molecular function go projects and 1 042 biologic process go projects; significant analysis showed that 4 cellular component go projects and 2 biologic process go projects were significantly enriched. These results indicated that the differentially expressed genes were involved in regulating extracellular domain (go:0044421 and go:0005576), lipoprotein complexes (go:0032994 and go:0034358) and inflammatory and traumatic reactions (go:0006954 and go:0009611). (4) KEGG pathway analysis showed that 576 differentially expressed genes were involved in 218 pathways, including metabolic pathways, transcriptional disorders in cancer, steroid hormone production. Biosynthesis, neurotrophic factor signaling pathway, complement cascade, coagulation cascade and cell adhesion molecule. significant analysis showed that only pentose and glucuronic acid conversion (ko00040) reached significant levels (qvalue 0.05). (5) 20 differentially expressed genes were randomly selected for fluorescence quantitative PCR verification, and the results showed that egr1, cpeb1, fgfr2, slc2a4 genes in SCNT The expression levels of hspb1, wnt2b, loc102279723, cdca7l, polr2a, crabp1, kifc3, cdc25a, ddit3, sdc1, loc102282421 genes in IVF group were significantly up-regulated, and were consistent with the RNA-seq data. (6) ivf-blastocyst and scnt-blastocyst predicted 1,435 and 1,499 new transcripts respectively. 23.62% and 22.28% of transcripts were predicted to have coding potential respectively. 3. Effects of histone methylation transferase inhibitors on embryonic development of yak SCNT (1) The morphology and cell viability of yak fibroblasts treated with low doses of gsk126 (10 and 20 microns) did not change significantly, and black spots appeared in cytoplasm of yak fibroblasts treated with high doses (40 and 80 microns). (2) After nuclear transplantation of yak fibroblasts treated with gsk126, it was found that the development rate of nuclear reconstituted blastocysts in low dose group, the number of te cells and the number of ICM cells in blastocysts were significantly higher than those in high dose group. According to the cell viability, blastocyst development rate and blastocyst development quality, the best effect of nuclear transplantation was obtained when the yak fibroblasts were treated with 20 micron gsk126 for 24 hours. (3) Histone methylation levels in IVF blastocysts, SCNT blastocysts and SCNT blastocysts treated with 20 micron GSK126 were analyzed. H3K27me2 and H3K27me3 histone methylation levels in nuclear transfer blastocysts were decreased, but H3K4me2 histone methylation levels were not significantly affected. (4) Analysis of embryo apoptosis and development-related genes, it was found that GSK126 treatment of blastocysts apoptotic gene BCL-2 mRNA expression level decreased, Caspase-9 mRNA expression level increased, and at the same time, it could up-regulate embryo development. Effects of NaF on the development and apoptosis of preimplantation embryos (1) After treated with NaF, the maturation rate, cleavage rate and blastocyst development rate of oocytes were significantly inhibited in a dose-dependent manner; ROS content in oocytes significantly increased and T-AO was inhibited after treated with high dose of NaF. The activities of C, GSH-Px, CAT and GSH induced oxidative stress in oocytes. (2) The activities of Caspase-3 and Caspase-9 in oocytes increased significantly with the increase of Na F dosage, the mRNA expression levels of apoptotic genes Bax, Caspase-3 and Caspase-9 increased, and the expression levels of anti-apoptotic genes Bcl-2 mRNA decreased. With the increase of NaF dosage, the total number of blastocysts and trophoblast cells and the number of inner cell mass decreased significantly, and the number of apoptotic cells in blastocysts increased. The mRNA expression of embryonic development-related genes Oct4, Nanog, Sox2 and CDx2 increased. The level of NaF decreased significantly with the increase of dose.
【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823.85
，
本文编号：2228810
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