Transcriptional activity and nucleolar ultrastructure of embryonic rabbit nuclei after transplantation to enucleated oocytes.

Changes in the level of transcriptional activity in 32-cell stage morula nuclei were studied after blastomere electrofusion to enucleated oocytes. Nuclear transplant recipients were pulse labelled with 3H-uridine during cultivation in vitro, embryos were then fixed and processed for autoradiography and electron microscopy. Transcriptional activity substantially decreased after 4.5 hr and was completely inhibited at last 15 hr after fusion. Transcription resumed thereafter in two-cell stage embryos and could be detected in both nuclei from 70% of the embryos analyzed. Transcription activity rapidly increased at the eight 16-cell stages, reaching the level typical for 32-cell stage nuclei used for the transfer. Changes in nucleolar ultrastructure after the nuclear transfer reflected the inhibition and subsequent reactivation of rRNA transcription. Nucleoli of 32-cell embryos had a typical structure of active nucleoli; many fibrillar centers surrounded and interconnected by threads of the dense fibrillar component and embedded in the granular component. Six hours following nuclear transplantation, these nucleoli underwent drastic changes including loss of granular material, collapse of nucleolar structure, and segregation of nucleolar components. Following the first cleavage, segregated fibrillar components of nucleoli manifested a complete inhibition of nucleolar transcription. Ribosomal RNA transcription was restored at the eight-cell stage and the sequence of ultrastructural changes was similar to that of the normal development. However, at the 32-cell stage, excessive extrusion of pre-ribosomal particles in the cytoplasm occurred, suggesting a possible alteration in regulating mechanisms of ribosome delivery. These results show that after fusion with enucleated metaphase II cytoplasm and subsequent activation, transcription is inhibited in donor embryonic nuclei and progressively increases again during cleavage; almost as in normal embryos. Migration of ribosomes into cytoplasm appears more intense in 32-cell stage reconstituted embryos but this does not seem to inhibit blastocyst building.

Cellular evaluation of bovine nuclear transfer embryos developed in vitro.

Cloned blastocysts developed in vitro for 7 d had a mean number of cells (82.86 +/- 5.35) as evaluated by nuclei counting in serial optical sections using confocal microscopy, after staining with propidium iodide. This number was not significantly different from that of control IVF embryos cultured under the same conditions during the same period (mean = 88.89 +/- 7.53). Semi-thin sections revealed that most of the blastocysts had an inner cell mass (10/12) and a blastocoele. Under transmission electron microscopy, the trophectoderm appeared well differentiated as a polarized epithelium with apical microvilli and lateral junctions including desmosomes with bound intermediate filaments. The cytoplasm sometimes contained immature mitochondria or a large number of residual bodies. About half of the blastocysts examined had a large amount of cellular debris in the perivitelline space or inside the blastocoele cavity. The cloned blastocysts were also able to hatch in vitro by day 8 and SEM indicated a normal morphology of the trophectoderm cells with numerous apical microvilli. The high number of excluded or degenerating cells found in some embryos may partially explain early embryonic mortality that follows transfer. However, these observations do not give a clear explanation for the high incidence of fetal losses.