Time-Dependent RNA Synthesis in Early Bovine Embryos Derived from In-Vitro Fertilization.

Abstract

Abstract. 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 µCi/ml of [5-3 H]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 weakened 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 3 H-uridine incorporation was analyzed by image analysis (NIH-157 analysis system). 3 H-Uridine incorporation into nuclei was observed in 43% of 2-cell embryos at 26 hpi although the level was low. The change of 3 Huridine 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 3 H-uridine into nucleoli was observed from 50 hpi. Twenty-two embryos at 5-cell to morula were incubated with labelled and unlabelled uridine as negative control showed no incorporation of 3 H-uridine. These results show that 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.