Remarkably, higher order chromatin reorganization continued beyond the 2 cell stage. New structures con taining centromeric and pericentromeric heterochromatin appeared at the 4 cell stage, forming structures very simi lar to classical chromocenters, i. e, a compact mass of peri centromeric heterochromatin surrounded by individual Ganetespib IC50 centromeres. During the same period, the number of nucleoli, which were now fully active, underwent an abrupt decrease between early 4 cell and late 4 cell stages. Finally, by the blastocyst stage, the overall nuclear organization was very similar to that of somatic cell nuclei in terms of nucleoli numbers and chromocenter organization. However, we scanned more than 1000 embryos in total, making the image analysis tedious.

In these condi tions, only the most obvious large scale nuclear move ments could be evaluated by visual analysis. We Inhibitors,Modulators,Libraries therefore configured semi automated image analysis tools Inhibitors,Modulators,Libraries particularly adapted to the size and geometry of the embryonic nuclei, describing quantitative morpho metric features of the nuclei and the NPBs/nucleoli. We also analyzed, in detail, heterochromatin behavior in the context of such morphological changes. Morphometric features of nuclei and NPBs/nucleoli DNA labeling was used to delineate the embryonic nu clei from the confocal 3D stacks and to calculate nuclear volumes. It should be mentioned that, for early stages, we distin guished early and late time points. However, at later stages, cellular divisions were no longer synchronous and such an analysis Inhibitors,Modulators,Libraries could not be performed. we thus pooled the data within each stage.

Figure 4 shows that the nuclear volume decreased progressively from the 2 cell stage to the blastocyst stage by a factor of 10, with the most marked decrease occurring between the 2 and 4 cell stages. We next performed a quantitative automated Inhibitors,Modulators,Libraries analysis of NPB/nucleolus numbers and volumes. As shown in Figure 5 and Table 4, the number of NPBs decreased slightly but significantly between the early and late 2 cell stage. This decrease during the 2 cell stage was accompanied by a marked modification in the distribution of NPB volumes the median value increased from 28. 7 um3 to 41. 5 um3. Interestingly, NPBs associated with pericentromeric heterochromatin were larger than those not associated with pericentro meric heterochromatin, both at early and late stages.

At 8 cell, the changes in nucleolar number Inhibitors,Modulators,Libraries and size distribution are much smaller, suggest ing that this fusion process selleck chemical is less prominent. In the following stage, the number of NPBs decreased drastically, as expected, from 11 NPBs in early 4 cell to 3 in late 4 cell embryos. Remarkably, the me dian value of the NPB volume reached 64. 5 um3 by the end of the 4 cell stage, suggesting that the number of NPBs decreases via NPB fusion.