Application of saponin on differential staining examination in animal blastocysts

Although there are several ways such as karyotyping to evaluate the quality and normality of embryos, the counting of total cell in blastocyst after the differential staining has been used as a simple indicator for quality of culture systems and normality of embryo itself. This differential staining method was regarded as a basic technique of early developmental biology of mammals, and it helps the scientific community to understand the signals regulatingmorphological events of early developmental process. The present study was undertaken to develop a simple and fast differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using saponin as a permeabilizing agent without using species-specific antibodies and complements. The prestained blastocyst with SYTO-13 (green) was exposed to saponin solution for propidium iodide (PI) permeation into TE cells and examined for the differential staining patterns. Three dimensional confocal microscopy was used to demonstrate the process of successful staining and showed the high impact on saponin treatment. Although the fluorescent images of blastocysts showed that one or two cell of TE stained to yellowish green, ICM was protected from saponin/PI mixture with the short exposure time of SYTO-13 pre-stained blastocysts. The total stainingprocedure did not exceed 30 min before examination under epi-fluorescence or confocal microscope. These results clearly demonstrate that saponin could be used as substituent molecule instead of species-specific antibodies and complements in differential staining examination for the first differentiation of mammalian embryos.