Curnow E C, Gunn I M, Trounson A O
Animal Gene Storage and Resource Centre of Australia, Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia.
Mol Reprod Dev. 2000 Jul;56(3):372-7. doi: 10.1002/1098-2795(200007)56:3<372::AID-MRD7>3.0.CO;2-W.
Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona-enclosed two-cell embryos (33-35 hr after initiation of sperm-egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% +/- 2.8%), cleavage (72.5% +/- 7.4%) and blastocyst development (56.1% +/- 6.4%) were achieved with the application of 1. 4 kV/cm as a single 100-microseconds pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50-microseconds pulses produced significantly less one-cell embryos (64.2% +/- 3.0%) compared to 1.4 kV/cm while cleavage (79.9% +/- 3.4) and blastocyst development (44.6% +/- 4.0%) were not different from that for unexposed control embryos (89.5% +/- 2.3% and 57.2% +/- 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30-microseconds pulse (69.7% +/- 5.7%) showed significantly lower cleavage (72.1% +/- 3.7%) and blastocyst rates (40.2% +/- 4.6%) compared to the unexposed control.
通过三种融合方案，对体外成熟并受精、带透明带的二细胞胚胎（精卵孵育开始后33 - 35小时）进行电融合，实验性地产生了四倍体牛囊胚。测试了1.0、1.4和2.4 kV/cm的场强，并分别测量了每种场强下的融合率、后续的卵裂率和囊胚发育率。施加1.4 kV/cm的单次100微秒脉冲可实现较高的融合率（76.5%±2.8%）、卵裂率（72.5%±7.4%）和囊胚发育率（56.1%±6.4%）。在刺激融合后30分钟和60分钟对胚胎进行评分。未观察到融合、卵裂或囊胚发育的时间效应。第7天囊胚的染色体制备显示，12.5%的融合胚胎为四倍体。这比未融合胚胎中自发四倍体未发生的情况有显著增加。与1.4 kV/cm相比，施加1.0 kV/cm的两个50微秒脉冲产生的单细胞胚胎显著减少（64.2%±3.0%），而卵裂率（79.9%±3.4）和囊胚发育率（44.6%±4.0%）与未处理的对照胚胎（分别为89.5%±2.3%和57.2%±3.2%）无差异。施加2.4 kV/cm的单次30微秒脉冲融合的胚胎（69.7%±5.7%）与未处理的对照相比，卵裂率（72.1%±3.7%）和囊胚率（40.2%±4.6%）显著降低。
