Microinjection of FITC-dextran into mouse blastomeres to assess topical effects of zona photoablation

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.