Nucleolar transcription was analysed in permeabilized pre-implantation mouse embryos at the four-cell, eight-cell, morula and early blastocyst stages using confocal microscopy to detect incorporated 5-bromouridine. The results demonstrated that the patterns of nucleolar transcription sites were common for all embryonic stages studied. They consisted most frequently of tightly associated groups of transcription foci similar to those encountered in somatic interphase cells. In addition, the nucleologenesis accompanying each cell cycle apparently gave rise to a different fluorescent pattern, that is to spatially separated fluorescent foci in the cells just after the resumption of rRNA synthesis. An immunoelectron microscopic analysis of the nucleolar transcription was also performed in the eight-cell embryos. A signal, usually consisting of clustered gold particles, was found specifically within nucleolar dense fibrillar components. This result was in agreement with established findings, which identify dense fibrillar component as the major site of nucleolar transcription in somatic cells.

Animals↗

PubMedDate not supplied

Chemically enucleated mouse oocytes: ultrastructure and kinetics of histone H1 kinase activity.

The objective of the study was to characterize the ultrastructure changes and biochemical mechanisms underlying the expulsion of the entire chromosome complement in chemically enucleated mouse oocytes. The ultrastructural studies demonstrated that the morphology of cytoplasts produced by etoposide-cycloheximide treatment were indistinguishable from intact metaphase I and II oocytes. Moreover, polar bodies formed by chemical enucleation were in almost all cases completely separated from the parent cytoplast and differed from normal polar bodies only in their chromatin content morphology and because they contained a slightly higher number of cytoplasmic organelles. The mode of polar body formation, however, in normal and chemically enucleated oocytes differs substantially: spindle involvement is important for normal polar body extrusion but plays no part in the protracted expulsion of chromosomes during chemical enucleation. After etoposide-cycloheximide treatment, histone H1 kinase activity remains high for the ensuing 6-8 h before declining gradually to basal levels 14 h after treatment. The expulsion of the polar body occurred only after the slowly declining H1 kinase activity reached basal levels. The activity of this kinase rose sharply to reach maximal levels within 4 h when the enucleated oocytes were removed from the inhibitor-supplemented medium and placed in normal medium. The findings in this paper indicate that cytoplasts produced by chemical enucleation are morphologically normal, thus suggesting that these enucleated cells are suitable for cloning studies. Although effective in mouse oocytes, we postulate that certain modifications to the enucleation technology are necessary before a reliable non-invasive protocol for ungulate oocytes will be available.

Animals↗

PubMedDate not supplied

Nuclear transplantation: reprogramming of transplanted nuclei.

Recent results in animal cloning have demonstrated that the events of cell differentiation can be reversed. This reversal necessarily requires genetic reprogramming of the transplanted nucleus. It has emerged from review articles that synthesis of nuclear and ribosomal RNA is blocked after fusion independently of cell cycle stage of the transferred nucleus, and that the cytoplasm condition of the cytoplast controls the speed of this inhibition. Transcription of the embryonic genome in nuclear transplant embryos begins either at the same stage as in normal development or even earlier. Expression analysis of individual genes showed up differences between nuclear transplant and normal embryos at the blastocyst stage. A certain degree of nuclear reprogramming after fusion is necessary, but exact timing and the qualitative sequence of the processes are probably not necessary for the successful embryonic development of the cloned embryo. In the future, observation of the expression of individual genes will bring more exact information about nucleus reprogramming.

Animals↗