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. © 1996 Wiley‐Liss, Inc.

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

“…The fact that only NT with RCCs directly resulted in production of a cloned rabbit suggests that reprogramming occurs more easily with this type of cells. Surprisingly, acH3K9/14 signals of in vivo fertilized rabbit embryos were enhanced at the 4-cell stage but not at the 8-cell stage at which the rabbit embryonic genome activation occurs (Kanka et al 1996, Henrion et al 2000, Brunet-Simon et al 2001. However, initiation of mRNA synthesis determined by in situ hybridization with a poly (U) probe was first detectable at the late 2-cell stage (Kanka et al 1993) and autoradiographically detectable initiation of transcription from an embryonic rabbit genome occurs at the 4-cell stage (Christians et al 1994).…”

The onset of foreign gene transcription in nuclear-transferred embryos of fish

“…Of course, whether in mammals [17] and amphibians [18] , or in fishes [19] , this kind of ratio was terribly low. The previous studies in mammals indicated that most donor nuclei could be remodelled and transcriptionally suppressed to a certain degree under the circumstance of egg cytoplasm, and commonly turned on their transcriptional activity at the moment similar to the timing of ZGA [2,16,20,21] . However, the result in fish nuclear transplantation is quite different.…”

“…In mammals, similar experiments have been done to study the changes of transcriptional activity of donor nuclei. Kanka et al [2] found that the transcriptional activity of donor nucleus derived from 32-cell stage morula substantially decreased after 4.5 h and was completely inhibited at last 15 h after the nuclear transfer. The transcription resumed thereafter in 2-cell stage embryos and rapidly increased at 16-cell stage, reaching the transcriptional level typically as that of the 32-cell stage nuclei used for the transfer.…”

Time-Dependent RNA Synthesis in Early Bovine Embryos Derived from In-Vitro Fertilization.

“…Recently, Kanka et al [24] observed an excessive extrusion of pre-ribosomal particles in the cytoplasm at the 32-cell stage of rabbit embryos after nuclear transplantation. So far, it is necessary to continue this kind of work to clarify the role of rRNA during the early development in mammals.…”