182 FAST STAINING METHODS FOR DIFFERENTIAL STAINING OF INNER CELL MASS AND TROPHECTODERM CELLS OF MAMMALIAN BLASTOCYSTS

Abstract

The present study was undertaken to develop a simple differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using the permeabilizing agent, saponin, without species-specific antibodies and complements. The nuclei of whole embryos were pre-stained to green by 5 ¼M SYTO 13 for 10 min. After washing, the green color of TE was turned to red by exposure to 100 ¼g/mL propidium iodide and 50 to 100 ¼g/mL saponin solution for 5 to 10 min. To confirm the exactness of staining patterns, the fluorescent nuclei of ICM and TE from mouse, pig, and bovine blastocysts were compared with 3D location by confocal microscopy. By the saponin mixture treatment method, in vitro-cultured mouse, pig, and bovine blastocysts were shown to have an ICM:TE ratio of 1:2.5, 1:4.5, and 1:3.6, with an average total cell number of 78 ± 14 (n = 45), 65 ± 18 (n = 49), and 150 ± 20 (n = 45), respectively. Although a few TE cells were stained to a yellowish-green color, the successful protection of the green color of ICM depended on the exposure time of blastocysts to the saponin mixture. The total time lapse of the procedure did not exceed 1 h. These results indicate that saponin could be used as a practical substitute for special antibodies and complements. So this differential staining for examining the ICM:TE ratio and the total cell count of mammalian blastocysts would be a fast and reliable method.