Collas P, Robl J M
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst 01003.
Biol Reprod. 1990 Nov;43(5):877-84. doi: 10.1095/biolreprod43.5.877.
Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p less than 0.005); they also fused at a higher rate (85% vs. 26%, p less than 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p less than 0.005), but was increased by using multiple pulses (85% vs. 68%, p less than 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p less than 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p less than 0.05) and development of oocytes to blastocysts (77% vs. 26%, p less than 0.001). Furthermore, 7.5 micrograms/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p less than 0.05) and development to blastocysts of manipulated embryos (46% vs. 11%, p less than 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p greater than 0.1).
对提高兔核移植效率的方法进行了评估。我们报告了受体卵母细胞年龄对核移植不同步骤的影响。研究了多次电脉冲以及融合后/激活培养基中的操作培养基和细胞松弛素B对激活和发育的影响。近期排卵的卵母细胞去核率（60%）高于老化卵母细胞（3%，p<0.005）；它们的融合率也更高（85%对26%，p<0.001）。与老化卵母细胞相比，新鲜排卵的卵母细胞激活率较低（分别为3%对37%；p<0.005），但通过多次电脉冲可提高激活率（85%对68%，p<0.05）。多次电脉冲也改善了囊胚发育率（48%对5%，p<0.001）。在含10%胎牛血清的碳酸氢盐缓冲培养基中对卵母细胞进行操作培养，也提高了激活率（100%对89%，p<0.05）以及卵母细胞发育至囊胚的比率（77%对26%，p<0.001）。此外，融合后/激活培养基中7.5微克/毫升的细胞松弛素B提高了激活率（78%对50%，p<0.05）以及操作后胚胎发育至囊胚的比率（46%对11%，p<0.001）。当应用上述改良方法时，总核移植胚胎（8至16细胞期供体细胞核）中有10%（23/230）或移植到受体的胚胎中有21%（2,3/110）发育为后代，这些比率与未操作的对照胚胎的发育比率相似（10%，4/41，p>0.1）。
