Nucleolus organizer regions and nucleoli in preattachment bovine embryos

Embryos (1-cell to elongated blastocyst stage) were recovered from superovulated heifers at surgery (Days 2-4; oestrus = Day 0), after slaughter (Day 4), or by transcervical flushing (Days 6, 7 and 14). The 175 embryos were cultured for 4, 8, 24 or 48 h, fixed on slides and sequentially stained with Giemsa and silver nitrate. Twenty-three 2-cell to blastocyst-stage embryos were fixed, embedded and examined by transmission electron microscopy. Argentophilic nucleolus organizer regions (Ag-NORs), indicative of transcriptionally active rRNA genes, were observed in embryos in which short- or long-term culture began at or after the late 8-cell stage. The nucleoli of embryonic cells also showed increased affinity for silver from the 8-cell stage onward. Differences in the number of Ag-NORs observed after the 8-cell stage reached statistical significance only when Day-5 and Day-7 embryos cultured for 4 h were compared. Ultrastructurally, the nucleoli were seen to develop from small, dense, fibrillar masses at the 2-cell stage, to ring-shaped structures (signifying a low level of activity) at the 8-cell stage. At the 16-cell stage the nucleoli became reticulated, suggesting an increase in activity, and by the morula and blastocyst stages they were characteristic of fully active nucleoli. It is concluded that a significant transcriptional activity of the rRNA genes in the embryos of cattle begins around the 8-cell stage.