RESUMEN
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.
Asunto(s)
Blastocisto/ultraestructura , Nucléolo Celular/ultraestructura , Región Organizadora del Nucléolo/ultraestructura , Animales , Bovinos , Células Cultivadas , Femenino , Metafase , Microscopía Electrónica , SuperovulaciónRESUMEN
In vivo nucleologenesis was studied in goat embryos from the pronuclear stage to the blastocyst stage by light and electron microscopy. Ultrastructural changes of the nucleoli were characterized by the following progression: homogeneous electron-dense fibrillar primary nucleoli in the pronucleus; small, dense fibrillar masses dispersed in clusters of chromatin at the two-cell stage; ring-shaped nucleoli made up of a fibrillar center surrounded by a layer of dense fibrillar components at the four-cell stage; reticulated nucleoli composed of a three-dimensional network of fibrillar components surrounded by small amounts of granular components at the eight-cell stage; fully developed compact-type nucleoli consisting of several fibrillar centers each surrounded by a layer of fibrillar components and abundant granular components in morulae and blastocysts. Moreover, it was concluded that activation of rRNA transcription, as evidenced by specific silver nitrate staining of the nucleolus organizer regions of metaphase chromosomes, occurs at the two- to four-cell stage and that the morphological changes accompanied a substantial increase in nucleolar transcriptional activity up to the blastocyst stage. This study provides evidence that a structure-function relationship exists during nucleologenesis in goat embryos.