Du Fuliang, Shen Perng-Chih, Xu Jie, Sung Li-Ying, Jeong B-Seon, Lucky Nedambale Tshimangadzo, Riesen John, Cindy Tian X, Cheng Winston T K, Lee Shan-Nan, Yang Xiangzhong
Department of Animal Science, Center for Regenerative Biology, University of Connecticut, 1390 Storrs Road, Storrs, 06269, USA.
Theriogenology. 2006 Feb;65(3):642-57. doi: 10.1016/j.theriogenology.2005.05.052. Epub 2005 Jul 19.
One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.
导致哺乳动物体细胞克隆效率低下的几个因素之一是小的体细胞供体细胞与大的受体卵母细胞之间融合不佳。本研究旨在测试植物血凝素（PHA）凝集活性对克隆牛胚胎融合率及后续发育潜力的影响。通过检查PHA对用不同剂量处理的孤雌生殖牛卵母细胞发育的影响（实验1）以及不同处理持续时间的影响（实验2）来确定PHA的毒性。选择PHA处理的有效剂量和持续时间（150微克/毫升，孵育20分钟），并用于比较体细胞核移植后的膜融合效率和胚胎发育（实验3）。还比较了使用体细胞供体成纤维细胞与卵丘细胞进行克隆，均进行了PHA处理（150微克/毫升，20分钟）和未进行PHA处理。经植物血凝素处理后，核供体成纤维细胞的融合率从33%提高到61%（P<0.05），卵丘细胞核供体的融合率从59%提高到88%（P<0.05）。对于成纤维细胞（13%对22%）和卵丘细胞（17%对34%；P<0.05），经PHA处理后每个使用的卵母细胞的核移植（NT）效率均得到提高。经PHA处理和未经PHA处理的克隆胚胎均进行玻璃化和胚胎移植测试，并获得了相似的存活率（约90%孵化）和妊娠率（17 - 25%）。这些胚胎经玻璃化和胚胎移植后出生了三头小牛；两头来自PHA处理组，一头来自非PHA对照组。我们得出结论，PHA处理显著提高了牛体细胞核移植的融合效率，因此增加了克隆囊胚的发育。此外，在确定的剂量和持续时间范围内，PHA对玻璃化后胚胎存活以及胚胎移植后的妊娠或产犊率均无不利影响。
