Li L, Munné S, Licciardi F, Neev J, Tadir Y, Berns M, Godke R, Cohen J
Animal Science Department, Louisiana State University, Baton Rouge 70803.
Zygote. 1993 Feb;1(1):43-8. doi: 10.1017/s0967199400001283.
The objective of the current experiments was to investigate whether all or only some blastomeres from precompacted mouse embryos were affected by zona photoablation. The microbeam of xenon chloride excimer laser (308 nm) was guided through an inverted microscope (non-contact system). Topical effects of lasing were determined by microinjection of a vital fluorescent dye of high molecular weight (fluorescein isothiocyanate [FITC] dextran) into the cell immediately adjacent to the site of zona photoablation. This dye is only passed onto daughter blastomeres and therefore allows study of specific cell lines. Embryonic growth was assessed following cell separation at the morula and blastocyst stage. Four-cell embryos treated with the laser had significantly fewer cells 12 h after zona photoablation than control embryos. A similar effect was noted after 24 h between dye injected embryos and those injected and lased simultaneously, indicating potential toxic effects of the laser treatment on the embryo. Effects on the blastomere closest to the site of ablation were evaluated by calculating the ratio of dyed cells to the total number of cells at specific time intervals. The ratios were similar in the dye and laser+dye groups of treated 4-cell embryos 36 h after treatment (0.22 and 0.23, respectively), indicating that the dye was still present in approximately 25% of the cells and that the negative effect of photoablation was evenly distributed among the blastomeres. It is concluded that zona photoablation may have long-term detrimental effects of a non-topical nature on precompacted mouse embryos in spite of the apparent precision of the laser spot size.
当前实验的目的是研究致密化前小鼠胚胎的所有卵裂球还是只有部分卵裂球会受到透明带光消融的影响。氯化氙准分子激光（308纳米）的微光束通过倒置显微镜（非接触系统）进行引导。通过将一种高分子量的活性荧光染料（异硫氰酸荧光素[FITC]葡聚糖）显微注射到紧邻透明带光消融部位的细胞中，来确定激光照射的局部效应。这种染料只会传递给子卵裂球，因此可以用于研究特定的细胞系。在桑椹胚和囊胚阶段进行细胞分离后，评估胚胎的生长情况。经激光处理的四细胞胚胎在透明带光消融后12小时的细胞数量明显少于对照胚胎。在24小时后，注射染料的胚胎与同时注射染料并接受激光照射的胚胎之间也观察到了类似的效应，这表明激光处理可能对胚胎具有潜在的毒性作用。通过计算特定时间间隔内染色细胞与细胞总数的比例，评估对最接近消融部位的卵裂球的影响。处理后36小时，处理过的四细胞胚胎的染料组和激光+染料组的比例相似（分别为0.22和0.23），这表明染料仍存在于约25%的细胞中，并且光消融的负面影响在卵裂球之间均匀分布。得出的结论是，尽管激光光斑尺寸看似精确，但透明带光消融可能对致密化前的小鼠胚胎具有非局部性质的长期有害影响。
