Sugimura Satoshi, Kobayashi Shuji, Hashiyada Yutaka, Ohtake Masaki, Kaneda Masahiro, Yamanouchi Tadayuki, Matsuda Hideo, Aikawa Yoshio, Watanabe Shinya, Nagai Takashi, Kobayashi Eiji, Konishi Kazuyuki, Imai Kei
National Livestock Breeding Center, Nishigo, Fukushima, Japan.
Cell Reprogram. 2012 Feb;14(1):29-37. doi: 10.1089/cell.2011.0060. Epub 2011 Dec 28.
We examined the influence of recipient oocytes on in vitro development, oxygen consumption, and gene expression in the resulting cloned bovine embryos. Oocytes derived from slaughterhouse ovaries and ovum pickup (OPU)-derived oocytes were used as recipient cytoplasts for the production of cloned embryos. A series of OPU sessions was conducted on Holstein cows without follicular growth treatment (FGT). In the same cows, we then performed dominant follicle ablation and subsequently administered follicle-stimulating hormone and prostaglandin F(2α) with controlled internal drug release device before a second series of OPU. Cumulus cells collected from single Holstein cows were used as donor cells. After measurement of oxygen consumption at the blastocyst stage with modified scanning electrochemical microscopy, analysis of 10 genes (CDX2, IFN-tau, PLAC8, OCT4, SOX2, NANOG, ATP5A1, GLUT1, AKR1B1, and IGF2R) was performed with real-time RT-PCR. Rates of fusion, cleavage, and blastocyst formation were not different among the treatment groups. Levels of oxygen consumption in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT were significantly lower than in blastocysts derived from artificial insemination (AI). However, oxygen consumption was increased in cloned blastocysts derived from OPU with FGT, depending on the individual oocyte donor. Furthermore, gene expression of IFN-tau and OCT4 in cloned blastocysts derived from OPU with FGT was similar to that in AI-derived blastocysts, whereas expression of those genes in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT was significantly different from that in AI-derived blastocysts. Thus, recipient oocytes collected by OPU in combination with manipulation of follicular growth in donor cows are suitable for producing cloned embryos.
我们研究了受体卵母细胞对所得克隆牛胚胎的体外发育、耗氧量和基因表达的影响。来自屠宰场卵巢的卵母细胞和经采卵（OPU）获得的卵母细胞被用作受体细胞质，用于生产克隆胚胎。在未经卵泡生长处理（FGT）的荷斯坦奶牛上进行了一系列OPU操作。然后，在同一批奶牛中，我们进行了优势卵泡消融，随后在第二轮OPU之前，通过可控性内部药物释放装置给予促卵泡激素和前列腺素F（2α）。从单头荷斯坦奶牛采集的卵丘细胞用作供体细胞。在使用改良的扫描电化学显微镜测量囊胚阶段的耗氧量后，采用实时RT-PCR对10个基因（CDX2、IFN-tau、PLAC8、OCT4、SOX2、NANOG、ATP5A1、GLUT1、AKR1B1和IGF2R）进行分析。各处理组之间的融合、分裂和囊胚形成率没有差异。来自屠宰场卵巢或未经FGT的OPU的克隆囊胚的耗氧量显著低于来自人工授精（AI）的囊胚。然而，根据单个卵母细胞供体的情况，来自经FGT的OPU的克隆囊胚的耗氧量有所增加。此外，来自经FGT的OPU的克隆囊胚中IFN-tau和OCT4的基因表达与来自AI的囊胚相似，而来自屠宰场卵巢或未经FGT的OPU的克隆囊胚中这些基因的表达与来自AI的囊胚有显著差异。因此，通过OPU采集受体卵母细胞并结合对供体奶牛卵泡生长的操作，适合用于生产克隆胚胎。
