Abstract
Rat preimplantation embryos were subjected to analysis of programmed SDS-electrophoresis with silver staining using Phast system (Pharmacia Co.Ltd.). Embryos in early development with ovulated ova, one cells, 2 cells, 4 cells, 8 cells, morulas, and early blastocysts were aspirated into 1 μ l capillary tube and kept at -60 °C until used. The embryos of each developmental stage were collapsed by repeating a temperature difference with dry ice and warm water (50 °C) several times and SDS-solution (1.5%) including 2-mercapotoethanol were aspirated into the tube, kept overnight, and applied to the electro-phoresis. By examining a minimum number of embryos per a lane (a capillary) for Phast gel to apply silver staining , five embryos were known to be the minimum number for the visual recognition of protein patterns and the number was standerized for each stage later on. The pattern of protein bands of preimplantation embryos development were scanned and calculated for molecular weight, relative percentage and protein content with Image Master (Pharmacia Co.Ltd). Typical bands of 50-52KD and 15-16kD were recognized in ovulated ova,1-cells, 2-cells, 4-cells, 8-cells, and morulas but the relative percent of the 2 bands was remarkably decreased in early blastocysts. When the protein patterns of serum, uterine fluid and granulosa cells of rat origin were compared with those of embryos, the band 48-51KD and 15-16KD was found to be specific for embryos. In addition, albumin,which is one of the main constituents of protein in serum, uterine fluid, and granulosa cells, was found to be remarkably less in the rat embryos. When zona pellucida originated from 6-11 ova were subjected to Phast system, no band was visible. Therefore, the pattern of protein band was considered to exist in substantial region of embryos.
