Time-dependent RNA synthesis in early bovine embryos derived from in-vitro fertilization.

摘要

1003 follicular oocytes were matured and fertilized in vitro, and subsequently cultured with a cumulus cell monolayer in TCM 199 with 5% fetal bovine serum. Embryos were transferred to TCM199 supplemented with 100 鍯i/ml of [5-3H]uridine at 26, 35,43, 50, 68 and 116 h post insemination (hpi), and cultured for 8 h at 39?C. Treated embryos were washed in phosphate-buffered saline, and the zona pellucida weakened with 5% proteinase K followed by fixation in acetic-alcohol. Preparations were air-dried, dipped in K5 emulsion, and exposed at 4? After a 4.5-day exposure, the preparations were developed in D19, and the density of 3H-uridine incorporation (as a measure of RNA synthesis) was determined by image analysis (NIH-157 analysis system). 3H-Uridine incorporation into nuclei was observed in 43% of 2-cell embryos at 26 hpi, although the level was low. The change of 3H-uridine incorporation level was not consistent until the 4-cell stage, and thereafter the level increased in a manner dependent ontime but not on cell number. Incorporation of 3H-uridine into nucleoli was observed from 50 hpi. Twenty-two embryos at the 5-cell to morula stages were incubated with labelled and unlabelled uridine as a negative control. They showed no incorporation of3H-uridine.