【作者】 吴侠；

【导师】 旭日干； 于海泉；

【作者基本信息】 内蒙古大学 ， 动物学， 2008， 博士

【摘要】 虽然体细胞克隆技术已经在多种动物中得以实现,但克隆胚胎怀孕率低、出生后异常等问题,严重制约了体细胞克隆技术的开发应用。有研究证实,供体细胞核在受体卵母细胞中不完全或异常的表观遗传重编程是导致克隆胚胎发育异常的重要原因。基因组DNA甲基化、组蛋白的共价修饰是重要的表观遗传修饰,二者具有复杂而广泛的生物学功能。在正常生殖细胞发生和胚胎发育过程中,染色体要经历广泛的DNA甲基化、去甲基化,核小体核心组蛋白也要通过各种共价修饰调节染色体功能,从而完成遗传信息的传递和调控胚胎的发育。目前,虽然已初步证实克隆胚胎存在DNA甲基化的异常,但组蛋白共价修饰是否也存在异常,还不是很清楚。为了深入探讨克隆胚胎组蛋白修饰的重编程,阐明胚胎的发育机理,进而改善克隆效率,本研究较系统的探讨了牛卵母细胞体外成熟、体外受精及胚胎体外发育过程中组蛋白修饰的动态变化,比较了体外受精胚胎与克隆胚胎早期发育过程中组蛋白修饰的差异。使用组蛋白去乙酰化酶抑制剂对供体细胞及受体卵母细胞进行了处理,检测了药物对供、受体表观遗传修饰以及克隆胚胎发育的影响,并对处理后供体核在去核卵母细胞中组蛋白修饰的重编程进行了分析。1.牛卵母细胞体外成熟及体外受精胚胎早期发育过程中组蛋白修饰的动态变化本研究探讨了组蛋白H3、H4不同甲基化、乙酰化修饰位点在牛卵母细胞体外成熟、体外受精及胚胎体外发育不同阶段的动态变化。结果显示,组蛋白H3、H4甲基化、乙酰化具有明显的阶段性变化。在卵母细胞成熟过程中,不同组蛋白不同修饰位点经历阶段性的、特异性去乙酰化,组蛋白甲基化则在卵母细胞成熟过程中没有明显变化。在精卵融合、精子核解凝集和重凝集过程中,乙酰化组蛋白H3、H4迅速定位于精子染色质,随后强的乙酰化信号定位于雄原核。与此同时,在卵母细胞核中,除H4K5ac,并没有检测到其他乙酰化信号;到染色质重新凝集时,检测到弱乙酰化信号(H3K9ac、H4K5ac、H4K8ac);而到雌原核形成时,乙酰化修饰加强。甲基化的H3K9me2和H3K4me3荧光信号在精子核膨大期和染色质再凝集期并没有出现;到原核形成时,雄性染色体组蛋白才逐渐被甲基化。与此同时,上述组蛋白甲基化信号在卵母细胞原核形成过程中维持了较高的水平。在随后的胚胎发育过程中,所研究的四种乙酰化组蛋白均存在于牛胚胎发育的各个时期,其中组蛋白H3K9ac,H3K18ac在8-细胞期明显减弱,到桑椹胚时增强。H4K8ac,H4K5ac在胚胎发育的不同阶段都具有较强的荧光信号,但在2-、4-和8-细胞期胚胎的间期卵裂球中,H4K8ac、H4K5ac定位于细胞核周边;而在分裂相的卵裂球中,H4K8ac、H4K5ac定位于凝聚染色体,且H4K5ac信号明显减弱。H3K18ac、H4K8ac、H4K5ac信号在囊胚滋胚层的染色较内细胞团强。与组蛋白乙酰化相比,甲基化H3K4在8-细胞期几乎完全消失,在随后的发育过程中,荧光信号有所恢复,而H3K9me2在8-细胞以后各期胚胎中则明显增强。上述结果表明,卵母细胞成熟有其特异的组蛋白修饰动态转变,组蛋白乙酰化逐渐被去除;受精是组蛋白甲基化、乙酰化逐渐恢复的过程,且不同修饰位点有其自身的动态变化特点;在胚胎发育过程中维持了组蛋白乙酰化、甲基化修饰。在受精及胚胎发育过程中,相同组蛋白的不同位点有相似的变化模式(H3K9ac vsH3K18ac;H4K8ac vs H4K5ac);功能相关的不同组蛋白修饰有其类似的动态变化(H3K9ac、H3K18ac vs H3K4me3),并且组蛋白乙酰化在牛胚胎细胞核中的定位也可能与合子基因组激活有关(H4K8ac、H4K5ac);而且组蛋白修饰与DNA甲基化密切相关(H3K9me2)。2.牛体外受精胚胎与体细胞克隆胚胎发育过程中组蛋白修饰的比较通过体细胞克隆技术构建重构胚胎并与体外受精来源的胚胎对组蛋白修饰的差别进行了比较。间接免疫荧光技术显示,从原核期到8-细胞期,克隆胚胎与体外受精胚胎存在着明显的组蛋白乙酰化、甲基化修饰的差异。高水平的H3K9ac、H3K18ac、H4K5ac、H4K8ac、H3K4me3和H3K9me2存在于8-细胞期之前的各期克隆胚胎中,并且H4K5ac和H4K8ac在克隆胚胎细胞核中的定位与体外受精胚胎也存在明显差异。8-细胞期以后,除了H3K9me2,其他组蛋白乙酰化、甲基化修饰的荧光强度及定位,在克隆胚胎都与体外受精胚胎类似。因此,克隆胚8-细胞前的发育阶段,存在着明显的组蛋白修饰异常,但当供体核基因组被激活,供体核组蛋白修饰经历较大程度的重编程,使得克隆胚胎与体外受精胚胎具有类似的组蛋白修饰模式。3.TSA处理转基因体细胞对克隆胚胎发育的影响Trichostatin A(TSA)作为特异的组蛋白去乙酰化酶抑制剂,能够增加细胞的组蛋白乙酰化水平,激活基因的表达,本试验使用TSA处理转基因供体细胞,并对基因组DNA甲基化水平、组蛋白乙酰化水平、报告基因的表达及克隆胚胎的发育进行了分析。结果表明转基因供体细胞对TSA存在明显的剂量效应,TSA浓度为100ng/mL时,产生明显的细胞毒性,大量细胞死亡(P＜0.01)。当TSA在较低浓度(5-50ng/mL)时,细胞形态发生改变,细胞增殖明显被抑制,S期细胞明显减少,细胞被抑制于G0/G1期。核型分析显示,TSA处理没有导致转基因细胞异常核型的形成。使用10-50ng/mL TSA处理供体细胞,明显增加了表达报告基因细胞的数量,基因组DNA甲基化水平降低,组蛋白乙酰化水平增加。使用经TSA处理的体细胞作为核供体进行核移植,获得了类似的卵裂、桑椹胚及囊胚率。当TSA浓度为50ng/mL时,抑制了桑椹胚(14.8%vs 27.3%、38.9%,P＜0.05),囊胚的发育(9.9%vs 20.7%、26.3%,P＜0.05)。转基因体细胞经TSA处理后,表达eGFP基因的囊胚数明显增加。结论:TSA处理供体细胞明显改变了转基因供体细胞的表观遗传特性,激活报告基因表达,报告基因表达的改变可以作为判定基因组DNA甲基化水平转变的标志。TSA处理供体细胞明显增加表达报告基因的囊胚数,但具有低水平DNA甲基化、高水平组蛋白乙酰化的转基因供体细胞并没有提高克隆胚胎的发育率。4.TSA处理卵母细胞对克隆胚胎发育及表观遗传重编程的影响本试验使用组蛋白去乙酰化酶抑制剂(TSA)处理卵母细胞,探讨其对卵母细胞组蛋白乙酰化、克隆胚胎发育及供体细胞染色质重编程的影响。结果表明卵母细胞成熟率与TSA浓度呈明显的剂量相关,较高浓度TSA(＞2.5ng/mL)处理,抑制了卵母细胞体外成熟,卵母细胞被抑制于MI期(10ng/mL、61.9%vs对照、31.4%,P＜0.05)。分析处理后卵母细胞核型显示,TSA(1、10ng/mL)处理没有明显影响卵母细胞的核型(85.5%、85.9%vs 81.8%,P＞0.05),且药物处理后明显增加了卵母细胞组蛋白乙酰化水平。TSA处理后的卵母细胞既促进了孤雌胚胎的发育(0.5ng/mL、28.7%,1ng/mL、36.4%,2.5ng/mL、25.9%,5ng/mL、27.1%vs对照、19.0%),也促进了克隆胚胎的发育(1ng/mL、39.3%vs对照、25.7%,P＜0.05),但是处理组与对照组囊胚细胞数没有明显差异。检测两种克隆胚胎1-细胞阶段的组蛋白修饰显示,供体细胞染色质在受体中经历去乙酰化及乙酰化重建。当供体细胞核发生早期染色体凝集时,对照和处理组H3K9ac、H3K18ac荧光信号消失;对照组供体染色体H4K8ac明显减弱,处理组该位点乙酰化消失;体细胞核H4K5ac信号在对照组卵母细胞中没有明显变化,处理组H4K5ac信号明显减弱。当重构胚被激活时,所用组蛋白乙酰化信号迅速恢复。TSA处理的卵母细胞还明显增加了供体染色质的稳定性(74.2%vs39.6%,P＜0.01)。结论:相对高浓度的TSA处理卵母细胞导致乙酰化水平明显升高,并且高的组蛋白乙酰化明显抑制了卵母细胞的成熟,但是TSA处理卵母细胞没有导致其染色体的异常。当将TSA处理后的卵母细胞用于孤雌胚胎和克隆胚胎的发育时,获得了高的孤雌和克隆囊胚率,并且TSA处理后的卵母细胞胞质明显促进了供体核的表观遗传重编程,并且增加了供体核在去核卵母细胞中的稳定性。更多还原

【Abstract】 Since the birth of Dolly, the first somatic cell nuclear transfer (SCNT) mammal, a number of species such as cattle, mouse, goat, pig, cat, mule, horse, rat, and ferret were produced. However, the low pregnant rate, high abortion occurrence, placental abnormalities, increased birth weight, and perinatal death resulted in a very low cloning efficiency. Differentiated somatic nuclei can be dedifferentiated in oocyte cytoplasm, converte to totipotent, and induce nuclear reprogramming, although the mechanisms involved are not clear. DNA methylation and histone modifications are considered to be importance in nuclear reprogramming. During natural reproduction, asymmetry demethylation, remethylaiton and histone modifications are observed between fertilization and blastocyst formation. Recently, more and more studies show that SCNT embryos exhibit defects during nuclear reprogramming. In this study, changes in histone modifications during bovine oocytes meiosis, fertilization, and embryos development in vitro were examined; histone modifications of embryos derived from IVF and SCNT were compared; Using trichostatin A (TSA), a histone deactylase inhibitor, to treat donor cells and recipient oocytes, change of DNA methylation and histone modifications of the resultant embryos were also investigated.1. Dynamic Changes in Histone Modifications during Bovine Oocytes Meiosis, in vitro Fertilization, and Embryo DevelopmentThis experiment was designed to investigate changes of histone methylation and acetylation during bovine oocytes meiosis, IVF, and embryo development. The results showed that histone acetylations were gradually disappeared from germinal vesicle (GV) stage to MII stage, histone methylations, however, did not change. At the time of sperm chromosome decondensation acetyl-histone H4 appeared. When sperm chromosome was recondensation, acetylation of histone H3 was observed. After male pronuclear formation, intensive histone acetylation signals were observed. When oocyte meiosis was activated, no histone acetylation signals were found, except for H4K5ac which expressed in AII-TII female chromosome. Intensive histone acetylation fluorescence signals were examined in female pronuclear. Methyl-histone H3 was not examined during male chromosome condensation and recondensation. Male pronuclear formed, and the histone methylation was examined. In female chromosome, intensive histone methylation fluorescence signals were examined from oocyte activation to pronuclear formation. When embryos cleaved, acetyl-histone H3 lysine 9 and lysine 18 (H3K9ac, H3K18ac) significantly reduced at 8-cell stage, and increased at morula stage. However, the fluorescence signals of acetyl-histone H4 lysine 8 and lysine 5 (H4K8ac, H4K5ac) did not change. Before zygotic genome activation, the enhanced staining for H4K8ac and H4K5ac were observed at the nuclear periphery. At mitosis, H4K8ac was located at condensing chromosomes, and acetylation signals of H4K5ac significantly reduced. At blastocyst stage, the H3K18ac, H4K8ac and H4K5ac signals were less intense in the inner cell mass (ICM) when compared to the trophectoderm cells (TE). During embryo development, fluorescence signals of trimethyl-histone H3 lysine 4 (H3K4me3) reduced or disappeared. However, the fluorescence intensity of dimethyl-histone H3 lysine 9 (H3K9me2) was gradually increasing during embryos development. At blastocyst stage, the levels of histone methylation between ICM and TE were with no difference. In conclusion, histone deacetylation was a meiotic-stage dependent and lysine residue-specific processes; During fertilization and embryo development, various lysine residues at the same histone had a similar dynamic changes (H3K9ac vs H3K18ac; H4K8ac vs H4K5ac); The similar dynamic changes of lysine residues were functional correlation (H3K9ac, H3K18ac vs H3K4me3); localizations of histone acetylation at nuclear stage was relative with zygotic genome activation (H4K8ac, H4K5ac), and changes of histone modifications were relative with embryonic DNA methylation (H3K9ac, H3K18ac, H3K4me3 vs H3K9me2).2. Comparative Study of Histone Modifications in Bovine IVF and SCNT EmbryosThe distribution patterns of acetylation on histone H3, H4, methylation on histone H3 lysine 9 and lysine 4 were examined in bovine preimplantation IVF and cloned embryos by using indirect immunofluorescence and scanning confocal microscopy. As results, high levels of histone acetylation and methylation were located in cloned embryos before donor genome activation (H3K9ac, H3K18ac, H4K5ac, H4K8ac, H3K4me3), and abnormal nuclear localizations of H4K8ac and H4K5ac were observed. Compared IVF with SCNT embryos, high level of H3K9me2 was examined during cloned embryos preimplantation development. When donor genome activation, all histone modifications were similar, except for H3K9me2, between IVF and cloned embryos. Therefore, somatic cells genome was widely epigenetic reprogrmming after somatic cell genome activation.3. Effect of Trichostatin A on Epigenetic Modifications of eGFP Transfected Cells and Subsequent Cloned Embryo DevelopmentBovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was in a dose dependent. When the TSA concentration was over 5ng/mL, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100ng/mL (P<0.01). Number of cells in S phase was significantly decreased in the 5 to 50ng/mL TSA-treated groups, while the majority of the cells were at G0/G1 phases. The number of eGFP expressed cells was approximately two-fold higher in 25ng/mL (30.5%) and in 50ng/mL (29.5%) TSA treatment groups when compared to the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50ng/mL TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50ng/mL TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, Treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation and eGFP gene expression was activated. The donor cells with reduced DNA methylation did not improve subsequent cloned embryo development. However, transgene expression was improved in cloned embryos.4. Treatment of oocyts with Trichostatin A, Resulted in Improved Nuclear Reprogramming and Cloned Embryo DevelopmentTrichostatin A (TSA), a histone deacetylase inhibitor, was used to treat bovine oocytes during in vitro maturation (IVM). Change of oocytes histone acetylation, cloned embryos development, and donor chromosome epigenetic reprogramming in enucleated oocytes was examined. The results showed that the effect of TSA on bovine oocytes was in a dose dependent. When the TSA concentration was over 2.5ng/mL, oocytes IVM were significantly inhibited. The majority of the oocytes inhibited at MI stage when TSA reached 10ng/mL (10ng/mL TSA, 61.9% vs control, 31.4%, P<0.05). However, effect of TSA on oocytes karyotype was not found (1ng/mL 85.5%, 10ng/mL 85.9% TSA vs 81.8% control, P>0.05, respectively). Histone acetylation levels of oocytes from TSA treatment were significantly increased. TSA treated oocytes were used to parthenogenetic development, and high development rates was achieved (0.5ng/mL 28.7%, 1ng/mL 36.4%, 2.5ng/mL 25.9%, 5ng/mL 27.1% vs controls 19.0%, respectively). Meanwhile, Transfer of donor cells to enucleated TSA-treated recipient oocytes resulted in a higher blastocyst development in lng/mL TSA (1ng/mL TSA 39.3% vs control 25.7%, P<0.05). However, similar total cell number per-blastocyst was observed.Recently, dynamic reprogramming of histone acetylation and methylation was investigated in the first cell cycle of cloned and TSA-treated cloned embryos. As results, when donor nuclear was induced premature chromosome condensation (PCC), part of somatic lysine acetylation on core histones (H3K9ac, H3K18ac) were quickly deacetylated in cloned and TSA-treated cloned embryos. The fluorescence intensity of H4K8ac was significantly decreased in control embryos, but the signals disappeared in cloned embryos from recipient oocytes treated by TSA. The H4K5ac fluorescence signals were not change in control embryos, and were significantly decreased in TSA treated group. Stability of donor chromosome was significantly increased when TSA treated oocytes were used as cytoplasm recipient (TSA-treated group 74.2% vscontrol 39.6%, P<0.01). In conclusion, bovine oocytes treated with TSA inceased global histione acetylation. Cloned embryos development and epigenetic reprogramming from TSA-treated oocytes were improved, and stability of donor chromosome in TSA-treated oocytes was increased.更多还原