Suteevun T, Smith S L, Muenthaisong S, Yang X, Parnpai R, Tian X C
Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Theriogenology. 2006 Jun;65(9):1704-15. doi: 10.1016/j.theriogenology.2005.09.015. Epub 2005 Oct 26.
The swamp buffalo (Bubalus bubalis) is a multi-purpose animal in agriculture that is challenged by extinction due to low reproductive efficiency. Nuclear transfer (NT) has been used to preserve special breeds of buffalo, as well as to increase the number of animals. However, cloned buffalo embryos have impaired development, as in other species. To understand the chromatin remodeling activities in cloned embryos and to improve NT technology, we examined the expression profiles of five genes involved in DNA and histone modifications, DNMT1, DNMT3A, DNMT3B, HAT1 and HDAC1, in single swamp buffalo metaphase II oocytes, NT and in vitro fertilized (IVF) embryos from the two-cell to the blastocyst stage, by quantitative real time RT-PCR. We observed similar expression dynamics for all genes studied in the NT and IVF embryos: relatively constant levels of expression for all genes were found from the MII oocyte up to the eight-cell stage; the levels of mRNA for HAT1 and DNMT3B continued to be stably expressed up to the blastocyst stage; while dramatic increases were seen for DNMT3A and HDAC1. Alternatively, the levels of DNMT1 started to decrease at the eight-cell stage. Despite the similarity in the dynamics of gene expression, dramatic differences in the relative levels of these genes between NT and IVF embryos were observed. The expression levels of all DNA modifying genes were higher in the NT embryos than in the IVF embryos at the eight-cell and blastocyst stages. The genes HDAC1 and HAT1 were also expressed significantly higher at the blastocyst stage in the NT embryos. Our results suggested differences in chromatin remodeling between NT and IVF embryos and that lower levels of DNA passive demethylation and higher levels of DNA de novo methylation occurred in the NT embryos. These observations are novel in the species of buffalo, and may be associated with developmental failure of cloned buffalo embryos due to the transcriptional repression effect of most genes studied here.
沼泽水牛（Bubalus bubalis）是农业中的一种多用途动物，但由于繁殖效率低下而面临灭绝的挑战。核移植（NT）已被用于保护特殊品种的水牛，以及增加动物数量。然而，与其他物种一样，克隆水牛胚胎的发育受到损害。为了了解克隆胚胎中的染色质重塑活动并改进核移植技术，我们通过定量实时RT-PCR检测了参与DNA和组蛋白修饰的五个基因DNMT1、DNMT3A、DNMT3B、HAT1和HDAC1在单个沼泽水牛中期II卵母细胞、核移植胚胎以及从二细胞到囊胚阶段的体外受精（IVF）胚胎中的表达谱。我们观察到在核移植胚胎和体外受精胚胎中研究的所有基因都有相似的表达动态：从MII卵母细胞到八细胞阶段，所有基因的表达水平相对恒定；HAT1和DNMT3B的mRNA水平在囊胚阶段一直稳定表达；而DNMT3A和HDAC1则显著增加。另外，DNMT1的水平在八细胞阶段开始下降。尽管基因表达动态相似，但在核移植胚胎和体外受精胚胎之间观察到这些基因相对水平的显著差异。在八细胞和囊胚阶段，核移植胚胎中所有DNA修饰基因的表达水平均高于体外受精胚胎。HDAC1和HAT1基因在核移植胚胎的囊胚阶段也有显著更高的表达。我们的结果表明核移植胚胎和体外受精胚胎之间在染色质重塑方面存在差异，并且在核移植胚胎中发生了较低水平的DNA被动去甲基化和较高水平的DNA从头甲基化。这些观察结果在水牛物种中是新颖的，并且可能与克隆水牛胚胎的发育失败有关，这是由于这里研究的大多数基因的转录抑制作用。
