Transcriptional activity in in vitro produced bovine two- and four-cell embryos

The objectives of this study on in vitro produced bovine two- and four-cell embryos were (1) to investigate the uptake of H-3-uridine through the plasma membrane, (2) to characterize the pattern of RNA synthesis during the second cell cycle, and (3) to measure the incorporation of H-3-uridine into de novo synthesized RNA. A total of 200 embryos were incubated with H-3-uridine for 15, 30, 60 (two- and four-cell embryos), 120 (four-cell embryos), 180 (two-cell embryos), and 240 min (two- and four-cell embryos), respectively. H-3-uridine uptake reached a maximum by 30 min in two-cell embryos, whereas four-cell embryos reached a maximum at 120 min. A total of 440 two-cell embryos were isolated 27-33 hr postinsemination (hpi), and 90 of these were incubated for 10 hr with H-3-uridine (200 mu Ci/ml). The remainder were incubated with H-3-uridine for 3 hr starting at 0-3 (n = 54), 3-6 (n = 75), 6-9 (n = 77), or 9-12 (n = 77) hr after cleavage to the two-cell stage. Control two-cell embryos (n = 67) were incubated with H-3-uridine supplemented with 5 mg/ml of unlabelled uridine for 10 hr (inhibition control), or they were incubated with H-3-uridine for 10 hr and RNase treated (100 mu g/ml) post fixation (RNase control). Subsequently, the embryos were processed for autoradiography. The long-term incubation revealed transcription (autoradiographically labelled nuclei) in a total of 77% of the two- and four-cell embryos. No transcription was observed in any of the 3 hr incubation groups. The RNase control embryos lacked labelling of the nuclei, whereas the inhibition control embryos only showed markedly reduced labelling. Finally, total RNA extraction was performed on a total of 336 two-cell embryos that were incubated with H-3-uridine or H-3-uridine supplemented with unlabelled uridine for 2, 5, or 10 hr. It was possible to detect an increasing amount of labelled RNA after the 2, 5, and 10 hr incubation periods, and it was possible to inhibit this incorporation competitively. Together the data demonstrate a low level of transcription during the second cell cycle without a well-defined transcriptional peak. (C) 1996 Wiley-Liss, Inc.