猪孤雌激活胚胎和卵母细胞体外成熟过程中的甲基化检测
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摘要
【目的】探索猪孤雌激活胚胎培养过程和猪卵母细胞体外成熟前后的DNA甲基化水平及其变化,为探讨猪孤雌激活胚胎和卵母细胞的发育提供理论依据。【方法】通过使用间接免疫荧光组化和激光共聚焦显微镜的荧光相对定量的两种方法来间接检测猪孤雌激活胚胎培养过程中和猪卵母细胞成熟前后的细胞5-甲基胞嘧啶的相对含量,观察其体外发育过程中甲基化水平的变化。【结果】随着体外培养时间的延长,猪孤雌激活胚胎的甲基化程度在二到桑葚胚时呈逐渐减弱趋势;在猪孤雌激活胚胎发育的过程中,同一个胚胎的不同卵裂球之间的甲基化水平有差异;内细胞团的甲基化水平低于滋养层。猪卵母细胞成熟前和体外成熟的基因组DNA甲基化水平相比,成熟前卵母细胞基因组DNA甲基化水平低于体外成熟后的卵母细胞基因组DNA甲基化水平;随着体外成熟时间的延长,猪卵母细胞的基因组DNA甲基化水平大致呈上升趋势。【结论】IVM/PA/IVC对猪卵母细胞及早期胚胎的甲基化模式有一定影响;猪卵母细胞体外培养过程中,猪卵母细胞核基因组的甲基化水平接近于正常体内的卵母细胞。
【Objective】To provide a theoretical basis for the development of parthenogenetic activated embryos and oocytes in pigs, the DNA methylation level and its changes before and after in vitro maturation of pig oocytes were explored.【Method】Indirect immunofluorescence histochemistry and relative fluorescence quantification of laser confocal microscopy were used to indirectly detect the relative content of 5-methylcytosine in the cells of porcine parthenogenetic embryo culture and before and after porcine oocyte maturation, and observe its in vitro development and changes of the level of methylation in the process.【Result】With the prolongation of in vitro culture time, the degree of methylation of parthenogenetic embryos in pigs was gradually reducing from the second to the morula; During the development of embryonic development in pigs, the level of methylation between different blastomeres of the same embryo was different; the level of methylation of the inner cell mass was lower than that of the trophoblast; Compared with the genomic DNA methylation level of mature oocytes before and after maturation, the genomic DNA methylation level of pre-mature oocytes was lower than the genomic DNA methylation level of oocytes after in vitro maturation; With the prolongation of time, the genomic DNA methylation level of porcine oocytes increased roughly.【Conclusion】IVM/PA/IVC had a certain effect on the methylation pattern of porcine oocytes and early embryos; During the in vitro culture of porcine oocytes, the methylation level of porcine oocyte nuclear genome was close to that of normal oocytes.
【Objective】To provide a theoretical basis for the development of parthenogenetic activated embryos and oocytes in pigs, the DNA methylation level and its changes before and after in vitro maturation of pig oocytes were explored.【Method】Indirect immunofluorescence histochemistry and relative fluorescence quantification of laser confocal microscopy were used to indirectly detect the relative content of 5-methylcytosine in the cells of porcine parthenogenetic embryo culture and before and after porcine oocyte maturation, and observe its in vitro development and changes of the level of methylation in the process.【Result】With the prolongation of in vitro culture time, the degree of methylation of parthenogenetic embryos in pigs was gradually reducing from the second to the morula; During the development of embryonic development in pigs, the level of methylation between different blastomeres of the same embryo was different; the level of methylation of the inner cell mass was lower than that of the trophoblast; Compared with the genomic DNA methylation level of mature oocytes before and after maturation, the genomic DNA methylation level of pre-mature oocytes was lower than the genomic DNA methylation level of oocytes after in vitro maturation; With the prolongation of time, the genomic DNA methylation level of porcine oocytes increased roughly.【Conclusion】IVM/PA/IVC had a certain effect on the methylation pattern of porcine oocytes and early embryos; During the in vitro culture of porcine oocytes, the methylation level of porcine oocyte nuclear genome was close to that of normal oocytes.
引文
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[13]Bao S Q,Obata Y,Carroll J,et al. Epigenetic modifications necessary for normal development are established during oocyte growth in mice[J]. Biology of Reproduction,2000,62(3):616-621.
[14]Ura K,Kurumizaka H,Dimitrov S,et al. Histone acetylation:Influence on transcription,nucleosome mobility and positioning,and linker histone-dependent transcriptional repression[J]. Embo Journal,1997,16(8):2096-2107.
[15]Kang Y K,Park J S,Koo D B,et al. Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos[J]. Embo Journal,2002,21(5):1092-1100.
[2]Shi W,Dirim F,Wolf E,et al. Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos[J]. Biology of Reproduction,2004,71(1):340-347.
[3]Mann M,Chung Y G,Nolen L D,et al. Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos[J]. Biology of Reproduction,2003,69(3):902-914.
[4]Tada M,Tada T,Lefebvre L,et al. Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells[J]. Embo Journal,1997,16(21):6510-6520.
[5]Rougier N,Bourc'His D,Gomes D M,et al. Chromosome methylation patterns during mammalian preimplantation development[J]. Genes & Development,1998,12(14):2108-2113.
[6]Fulka H,Mrazek M,Tepla O,et al. DNA methylation pattern in human zygotes and developing embryos[J]. Reproduction,2004,128(6):703-708.
[7]舒金辉. 水牛体细胞核移植与甲基化检测的研究[D]. 南宁:广西大学,2007.
[8]Kono T,Obata Y,Wu Q L,et al. Birth of parthenogenetic mice that can develop to adulthood[J]. Nature,2004,428(6985):860-864.
[9]Dean W,Santos F,Stojkovic M,et al. Conservation of methylation reprogramming in mammalian development:Aberrant reprogramming in cloned embryos[J]. Proceedings of the National Academy of Sciences of the United States of America,2001,98(24):13734-13738.
[10]Hiura H,Obata Y,Komiyama J,et al. Oocyte growth-dependent progression of maternal imprinting in mice[J]. Genes to Cells,2006,11(4):353-361.
[11]Santos F,Hendrich B,Reik W,et al. Dynamic reprogramming of DNA methylation in the early mouse embryo[J]. Developmental Biology,2002,241(1):172-182.
[12]Shi W,Haaf T. Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure[J]. Molecular Reproduction and Development,2002,63(3):329-334.
[13]Bao S Q,Obata Y,Carroll J,et al. Epigenetic modifications necessary for normal development are established during oocyte growth in mice[J]. Biology of Reproduction,2000,62(3):616-621.
[14]Ura K,Kurumizaka H,Dimitrov S,et al. Histone acetylation:Influence on transcription,nucleosome mobility and positioning,and linker histone-dependent transcriptional repression[J]. Embo Journal,1997,16(8):2096-2107.
[15]Kang Y K,Park J S,Koo D B,et al. Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos[J]. Embo Journal,2002,21(5):1092-1100.