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 moru la nuclei were studied after blastomere electrofusion to enucleated oo cytes. Nuclear transplant recipients were pulse labelled with H-3-urid ine 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 emb ryos analyzed. Transcription activity rapidly increased at the eight 1 6-cell stages, reaching the level typical for 32-cell stage nuclei use d for the transfer. Changes in nucleolar ultrastructure after the nucl ear transfer reflected the inhibition and subsequent reactivation of r RNA transcription. Nucleoli of 32-cell embryos had a typical structure of active nucleoli; many fibrillar centers surrounded and interconnec ted by threads of the dense fibrillar component and embedded in the gr anular component. Six hours following nuclear transplantation, these n ucleoli underwent drastic changes including loss of granular material, collapse of nucleolar structure, and segregation of nucleolar compone nts. 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 t he sequence of ultrastructural changes was similar to that of the norm al development. However, at the 32-cell stage, excessive extrusion of pre-ribosomal particles in the cytoplasm occurred, suggesting a possib le alteration in regulating mechanisms of ribosome delivery. These res ults 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 in tense in 32-cell stage reconstituted embryos but this does not seem to inhibit blastocyst building. (C) 1996 Wiley-Liss, Inc.