Ferreira Christina Ramires, Meirelles Flávio Vieira, Yamazaki Walt, Chiaratti Marcos Roberto, Méo Simone Cristina, Perecin Felipe, Smith Lawrence Charles, Garcia Joaquim Mansano
Departamento de Medicina Veterinária Preventiva e Reprodução Animal, FCAV-UNESP Jaboticabal-SP, Brazil.
Cloning Stem Cells. 2007 Winter;9(4):618-29. doi: 10.1089/clo.2006.0082.
The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.
克隆动物中控制供体细胞来源的线粒体DNA（mtDNA）结果的机制在很大程度上仍然未知。本研究旨在调查重构牛胚胎在植入前发育过程中以及克隆动物中体细胞和胚胎mtDNA的动力学。该实验涉及两种不同的胚胎重构程序，并在胚胎发育的五个不同阶段对其进行评估，以测量供体细胞mtDNA（印度瘤牛）的比例，以及该mtDNA在卵裂过程中的分离情况。使用等位基因特异性PCR和荧光染色的特异性引物，对每个采样的卵裂球、整个囊胚以及成纤维细胞来源的新生克隆动物的组织中供体细胞（印度瘤牛）与宿主卵母细胞（黄牛）mtDNA的比例（异质性）进行了估计。尽管在9-16细胞阶段显著下降（5.8%；p<0.05），但NT-B受精卵和囊胚显示出相似的异质性水平（分别为11.0%和14.0%）。然而，NT-F重构受精卵中的异质性水平从初始的低水平（4.7%）增加到9-16细胞阶段的12.9%（p<0.05）。NT-F囊胚含有低水平的异质性（2.2%），并且在克隆的新生小牛的配子或组织中未检测到体细胞来源的mtDNA。这些结果表明，与卵裂球的mtDNA不同，体细胞的mtDNA在卵裂过程中要么进行复制，要么逃避降解，尽管在囊胚阶段之后它会被降解，或者在体细胞发育过程中丢失，这从出生时缺乏供体细胞mtDNA可以看出。
