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

Abstract

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.

Early and delayed aspects of nuclear reprogramming during cloning

“…Increased detection of newly synthesized heterogeneous nuclear RNA (hnRNA) is also observed in cloned bovine embryos (Lavoir et al, 1997). Ultrastructural analysis of bovine nuclear transfer embryos revealed widespread transcriptional silencing following nuclear transfer, and then transcriptional re‐activation much like normal embryos, but with an excess production of ribosomes (Kanka et al, 1996). Remodelling of the nucleolus in bovine clones has been reported as being more efficient using activated oocytes as recipients as compared with non‐activated oocytes (Kanka et al, 1999).…”

Cloning: questions answered and unsolved

“…The molecular basis of reprogramming during cloning is entirely unknown. Although numerous correlates have been examined, such as effects on nuclear matrix proteins (Moreira et al, 2003), nuclear envelope components (Kubiak et al, 1991), nucleolar components (Kanka et al, 1996), and histone proteins (Gao et al, 2004a, and references therein), the actual factors that are responsible for guiding the specific repression and activation of genes in the cloned embryo have not been identified. Additionally, it is clear that reprogramming is only initiated in the oocyte, and then continues during cleavage and subsequent development, raising the question of what reprogramming factors exist in the oocyte, and what specific changes must occur in the oocyte to set the stage for continued reprogramming later.…”