Time-dependent RNA synthesis in early bovine embryos derived from in-vitro fertilization
1997
Iwasaki, S. (Roslin Inst., Midlothian (UK)) | Wilmut, I. | Campbell, K.H.S.
RNA synthesis in bovine embryos produced in vitro was investigated to clarify changes in transcriptional activity. Overall 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 (FBS). Embryos were transferred to TCM199 supplemented with 100 mu-Ci/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 PBS and the zona pellucida weakered with 5% proteinase K followed by fixation in acetic-alcohol. Preparations were air-dried, dipped in K5 emulsion, and exposed at 4 C. After 4.5 days exposure, the preparations were developed in D19 and the density of 3H-uridine incorporation was analyzed 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 4-cell stage and thereafter the level increased in a time-dependent manner but not dependent on cell number. Incorporation of 3H-uridine into nucleoli was observed from 50 hpi. Twenty-two embryos at 5-cell to morula were incubated with labeled and unlabeled uridine as negative control showed no incorporation of 3H-uridine. These results show the the transcription of early bovine embryos fertilized in vitro is already active in 2-cell embryos at 26 hpi, the level increases in a time-dependent manner after 5-cell stage and is not dependent on the cell number
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