We also developed new image analysis tools to quantify various nuclear parameters of the 3D FISH images, i. e, the nu clear volume, the number of NPBs/nucleoli, the nuclear polarity, the number and shape of pericentromeric het erochromatin structures, and their proximity to NPBs/ nucleoli. selleck compound Our results highlight differences in nuclear organization in paternal and maternal inherited genomes at the 1 cell stage. We also find that the reprogramming of the embryonic genome, which starts at the Inhibitors,Modulators,Libraries 2 cell stage, under goes several abrupt changes during preimplantation development. Results Unique nuclear organization of zygotes We first analyzed the distribution of centromeric and pericentromeric hetero chromatin in zygotes throughout the first cell cycle after fertilization.
At that stage, the parental genomes are separated in two haploid pronuclei containing nonfunctional Inhibitors,Modulators,Libraries NPBs, and zygotes can be clas sified in substages from PN0 to PN5. As previ ously described in the literature, we observed markedly different reorganizations within the male and female pronuclei from PN0 to PN5. Just after fertilization, pericentromeres organized Inhibitors,Modulators,Libraries rapidly around the NPBs in the female pronucleus whereas in the male pronucleus, they remained associated together in more or less unorganized masses located in the center. Remarkably, at PN3, only 3% of the NPBs were not associated with pericentro meric signals in the fPN as opposed to almost 30% in the mPN. We also noticed that the number of NPBs, while decreasing with time in both PNs, remained approximately twice as high in the mPN as compared to the fPN.
It was only in the late 1 cell stage that pericentromeric heterochromatin adopted the same dis tribution in mPN and fPN, namely, more or less complete shells around the NPBs, in which the minor satellite centromeric signals were embedded. Pericentromeric heterochromatin was also found at the nuclear per iphery, in association with centromeric Inhibitors,Modulators,Libraries spots. Pericentromeric heterochroma tin formed other remarkable features such as beaded filaments extending from the nucleolar periphery to wards the nuclear periphery. In addition, as in earlier substages, the number of NPBs remained lower in the fPNs than in the mPNs. Owing to the tight association we observed between pericentromeric heterochromatin and the NPBs, we next analyzed the localization of rDNA sequences also known to be structurally associated with NPBs.
For that purpose, we performed a dual FISH with major satellite and rDNA probes. We found most of the rDNA signals Inhibitors,Modulators,Libraries associated with pericentromeric signals at the periphery of the NPBs or within the pericentromeric CHIR99021 solubility fila ments. We sometimes noticed rDNA signals joining two NPBs. More surpris ingly, we frequently observed rDNA foci at the nuclear periphery, associated with pericentromeric signals. In fact, 80% of the pericentro meric signals at the nuclear periphery were flanked by rDNA foci.