Local and global changes in the morphology and distribution of replication centres in rapidly expanding nuclei

Sperm pronuclei assembled in Xenopus egg extracts undergo a 10-fold increase in volume over a typical 150-min incubation. Double immunofluorescence with anti-proliferating cell nuclear antigen (PCNA) antibodies and biotinylated anologues of thymidine can be used to distinguish different phases of S-phase. The mean diameter of pronuclei at initiation was 12.42 microns. During the elongation phase of S-phase the mean diameter increased to 17.44 microns, equal to a sevenfold increase in nuclear volume. Confocal microscopy was used to investigate the morphology and distribution of replication centres through S-phase. In early S-phase nuclei, the mean centre-to-centre distance between any replication centre and its 10 nearest neighbours was 1.24 microns; this decreased significantly to 0.90 micron in mid S-phase nuclei and 0.99 micron in late S-phase nuclei. In addition, the morphology of replication foci also changed. In early S-phase nuclei, each replication focus was a discrete entity. However, as S-phase progressed neighbouring replication foci appeared to fuse giving rise to ring-like structures or string-like structures. Three-dimensional reconstructed images of z-series collected through early and late S-phase nuclei indicated that as nuclei enlarge replication centres appear to accumulate at the nuclear periphery. A model is proposed to accommodate the results described below.