牛体外受精胚胎及克隆胚胎发育过程中组蛋白修饰表观遗传-…培训讲学

作者姓名：吴侠论文题目：牛胚胎发育过程组蛋白修饰及克隆胚胎表观遗传重编程的作者简介：吴侠，男，1980年09月出生，2003年07月师从于内蒙古大学旭日干教授，于2008年07月获博士（硕士）学位。

随着十年前第一只体细胞克隆哺乳动物的诞生，近年来，体细胞核移植技术已经在其他物种中得到实现，且体细胞核移植技术已经越过了种属的界限。

但就目前的技术水平而言，体细胞克隆胚胎怀孕率低、出生后胎儿异常的问题依然没有解决，这些问题的存在严重制约了体细胞克隆技术的广泛应用。

许多研究证实，供体细胞核在受体卵母细胞中的不完全或异常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。

正常生殖细胞发生及胚胎发育过程，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，对于克隆胚胎重编程机理的研究还处于初级阶段，虽然证实克隆胚胎存在DNA甲基化的异常，但是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，进而改善克隆效率，阐明胚胎的发育机理，本研究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. 牛卵母细胞减数分裂、体外受精及胚胎体外发育过程中组蛋白修饰动态变化的研究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。

牛卵母细胞成熟过程不同组蛋白不同修饰位点经历阶段的，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。

精卵融合，精子核解凝集，重凝集，乙酰化组蛋白H3、H4迅速定位于雄性染色体，随后强的乙酰化信号定位于雄原核。

卵母细胞恢复减数分裂，排出第二极体，除H4K5ac，没有检测到其他乙酰化信号，染色质凝集，检测到弱乙酰化信号（H3K9ac、H4K5ac、H4K8ac），随后，雌原核也表现为强的乙酰化修饰。

精子核膨大和再凝集，没有检测到H3K9me2，H3K4me3荧光信号，原核形成，雄性染色体组蛋白逐渐被甲基化。

卵母细胞减数分裂恢复及原核形成过程维持了高的组蛋白甲基化水平。

胚胎发育，乙酰化的组蛋白存在于牛胚胎发育的各个时期，组蛋白H3K9ac，H3K18ac在8细胞期明显减弱，桑葚胚增强。

H4K8ac，H4K5ac在胚胎发育的不同阶段都具有较强的荧光信号。

合子基因组激活前，细胞间期，H4K8ac、H4K5ac定位于细胞核周边，有丝分裂期，H4K8ac定位于凝聚染色体末端，H4K5ac信号明显减弱。

H3K18ac、H4K8ac、H4K5ac在囊胚滋胚层的染色较内细胞团强。

与组蛋白乙酰化相比，H3K4me3在合子基因组激活后几乎完全消失，而H3K9me2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；胚胎发育过程维持了组蛋白乙酰化、甲基化修饰，受精及胚胎发育过程中，相同组蛋白的不同位点有相似的调控模式（H3K9ac vs H3K18ac；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细胞核中的定位与合子基因组激活有关（H4K8ac、H4K5ac）；组蛋白修饰与DNA甲基化密切相关（H3K9ac、H3K18ac、H3K4me3 vs H3K9me2）。

2. 牛体细胞核移植系统的优化及体外受精与核移植胚胎发育过程组蛋白修饰的比较本研究建立了成体海福特公牛耳皮肤成纤维细胞系，对其体外生长特性及核型进行了分析。

通过核移植技术构建重构胚，并比较与体外受精来源的胚胎在发育过程中组蛋白修饰的差别。

结果表明成体成纤维细胞体外生长具有正常的形态、分裂增殖特性和染色体数目。

为了提高重构卵的融合率，本研究对显微操作后不同恢复时间的重构卵实施电融合，结果恢复0.5hr融合率明显高于恢复 1.5hr （73.4% vs 52.7%，P<0.01）。

利用间接免疫荧光技术比较体外受精和克隆胚胎发育不同阶段，组蛋白不同修饰位点乙酰化、甲基化的动态变化差异显示，供体基因组激活前，克隆胚胎与体外受精胚胎相比，存在明显的组蛋白乙酰化、甲基化修饰差异，高水平的H3K9ac、H3K18ac、H4K5ac、H4K8ac、H3K4me3、H3K9me2存在于8细胞前各发育阶段，并且重构胚H4K5ac、H4K8ac在细胞核中的定位与体外受精胚胎存在明显差异。

基因组被激活，除了H3K9me2，克隆胚所有组蛋白乙酰化、甲基化修饰的荧光强度及定位都与体外受精胚胎类似。

因此，克隆胚胎发育过程中明显的组蛋白重编程异常。

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，能够增加细胞组蛋白乙酰化水平，激活基因的表达，本研究使用组蛋白TSA处理转基因供体细胞，并对基因组DNA甲基化、组蛋白乙酰化、报告基因表达及克隆胚胎发育进行分析。

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）。

TSA在较低浓度（5-50ng/mL），细胞形态发生改变，细胞增殖明显抑制，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甲基化水平将低，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、桑葚胚及囊胚率。

TSA浓度为50ng/mL时，抑制了桑葚胚（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DNA甲基化、高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研究本研究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浓度呈现明显的剂量相关，较高浓度TSA（>2.5ng/mL）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正常率显示，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）。

药物处理后明显增加了卵母细胞组蛋白乙酰化水平。

TSA处理后的卵母细胞即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），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，供体细胞核发生早期染色体凝集，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 在对照组卵中没有明显变化，处理组H4K5ac明显减弱。

TSA处理的卵母细胞明显增加了供体染色质的稳定性（74.2% vs 39.6%，P<0.01）。

因此，药物处理后的卵母细胞促进了体细胞核的重编程，且增加了供体核在卵母细胞中的稳定性。

关键词：牛卵母细胞体外成熟，体外受精及胚胎发育，体细胞核移植，Trichostatin A，组蛋白修饰Histone Modifications and Epigenetic Reprogramming in Bovine IVF and Cloned Embryos DevelopmentABSTRACTTen years ago, the first somatic cell nuclear transfer (SCNT) mammal was producted. The birth of Dolly sparked media frenzy and a prolonged ethical debate. From then on, a great deal of cloned mammals was production in various species, and the technology had surpassed limitation of species. However, the effects of thesetechnologies on the development of reconstructed embryos remain largely uncontrolled in all species so far successfully cloned. High abortion rates, placental abnormalities, increased birth weight, and perinatal death have been reported in several species. These disfigurements of cloned embryos development result it was impossible to produce animal widely. Differentiated somatic nuclei can be dedifferentiated in oocyte cytoplasm, converted to a totipotent stage, and induced somatic gene expression, a process termed nuclear reprogramming. How to reprogramming of somatic nuclei in enucleated oocyte was unclear. DNA methylation and histone modifications were considered that it is importance in nuclear reprogramming. During natural reproduction, asymmetry demethylation, remethylaiton and histone modification were observed between fertilization and formation of the blastocyst. Recently, more and more studies show that cloned 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 were compared IVF with SCNT during bovine embryos development. Donor cells and oocytes were treated with Trichostatin A (TSA), a histone deactylase inhibitor. Change of DNA methylation and histone modifications after TSA treatment were analyzed. Cloned embryos generated from TSA-treated donor and receptor cells, and epigentic reprogramming was also investigated.1. Dynamic changes in histone modifications during bovine oocytes meiosis, in vitro fertilization, and embryos developmentIn this study, change of histone methylation and acetylation during bovine oocytes meiosis, IVF, and embryos development was investigated. Histone acetylations were gradually disappeared, and histone methylations were not change during bovine meiotic maturation. When sperm fused with oocytes during fertilization, sperm chromosome was condensed, recondensed. Oocytes meiosis was resumption, Pb2 was expelled, and chromosome was recondensed. At last, male and female pronuclear formed. Acetyl-histone H4 was observed in decondensed sperm immediately. When sperm chromosome was recondensation, acetylation of histone H3 was found, intensive histone acetylation signals were observed in male pronuclear. When oocytes meiosis was activated, no histone acetylation signals were found, except for H4K5ac, in 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 oocytes acetivation to pronuclear formation. When embryos were cleavage, acetyl-histone H3 lysine 9 and lysine 18 (H3K9ac, H3K18ac) were significantly reduce at 8-cell stage, and increased in morula. However, the fluorescence signals of acetyl-histone H4 lysine 8 and lysine 5 (H4K8ac, H4K5ac) were 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 terminal chromosome, and acetylation signals of H4K5ac was significantly reduce. At blastocyst stage, the staining of H3K18ac, H4K8ac and H4K5ac signals were less intense in the inner cell mass (ICM) when compared to the trophectoderm cell (TE). During embryos development, fluorescence signals of trimethyl-histone H3 lysine 4 (H3K4me3) was disappeared or reduced. However, the fluorescence intensity of dimethyl-histone H3 lysine 9 (H3K9me2) was gradually increasing during embryos development. At blastocyst stage, the different levels of histone methylation between ICM and TE were not evident. In conclusion, the results showed that histone deacetylation was a meiosis stage dependent and lysine residue-specific process; During IVF and embryos 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 found that were functional correlation (H3K9ac, H3K18ac vs H3K4me3); Locations of histone acetylation at nuclear was relative with zygotic genome activation (H4K8ac, H4K5ac), and change of histone modifications were relative with embryonic DNA methylation during embryos development (H3K9ac, H3K18ac, H3K4me3 vs H3K9me2).2. Optimization of SCNT technology and comparison of histone modifications inIVF and cloned bovine embryosBovine ear fibroblast cell line was derived from surgical excision biopsy performed on a 6 years older Hereford. The fibroblast cells were chosen for further morphological observation, growth mensuration and karyotyping. The results indicated that the cellspossession normal morphology, proliferation characteristics and chromosome number. Fusion rates of reconstructed embryos was significantly different between recovered 0.5hr and 1.5hr after micromanipulation (73.4% vs 52.7%，P<0.01). The 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 locations 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 Cellsand Subsequent Cloned Embryo Development in BovineBovine 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 5 ng/ml cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P<0.01). Number of cells in S phase was significantly decreased in the 5 to 50 ng/ml TSA-treated groups, while the majority of the cells were at G0/G1 phases. The number of eGFP-expressed cells were approximately two-fold higher in 25 ng/ml (30.5%) and 50 ng/ml (29.5%) TSA 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 50 ng/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 50 ng/ml TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher thanthat 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. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development. However, transgene expression was improved in cloned embryos.4. Treatment oocyts with A Epigenetic Modifier, Trichostatin A, Results inImproved Cloned Embryos Development and Reprogramming in BovineTrichostatin 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 2.5 ng/mL, oocytes IVM was significantly inhibited. The majority of the oocytess inhibited at MI stage when TSA reached 10 ng/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 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 control、19.0%, respectively). Meanwhile, Transfer of donor cells to TSA-treated recipient oocytes resulted in high blastocyst development in 1 ng/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 wassignificantly increased when TSA treated oocytes were used as cytoplasm recipient (TSA-treated group 74.2% vs control 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.Key words: bovine oocytes in vitro maturation, IVF and embryo development, SCNT, Trichostatin A, histone modifications。