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Laurence Gall, Daniel Le Bourhis, Sylvie Ruffini, Claire Boulesteix, and Xavier Vignon

It is clear from a wide range of studies that the nuclear/cytoplasmic distribution of Cdc25C has important functional consequences for cell cycle control. It is now admitted that in somatic cells, the localization of Cdc25C in the cytoplasm is required to maintain the cell in an interphasic state and that Cdc25C has to translocate to the nucleus just before M-phase to induce mitotic events. We characterized the expression and localization of Cdc25C during oocyte maturation, the first embryo mitosis, and the first steps of somatic cell nuclear transfer (SCNT) in cattle. We demonstrated that Cdc25C was expressed throughout the maturation process and the early development. We clearly showed that Cdc25C was localized in the nucleus at the germinal vesicle stage and during the early development until the blastocyst stage. However, the signal change in blastocyst and Cdc25C became cytoplasmic as is the case in somatic cells. Thus, oocytes and early embryonic cells presented a specific nuclear Cdc25C localization different from the one observed in somatic cells, suggesting that Cdc25C could have a particular localization/regulation in undifferentiated cells. Following SCNT, Cdc25C became nuclear as soon as the nucleus swelled, and this localization persisted until the blastocyst stage, as is the case in in vitro fertilized embryos. The Cdc25C nuclear localization appeared to constitute a major change, which could be associated with the reorganization of the somatic nucleus upon nuclear transfer.

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Andrey Pichugin, Daniel Le Bourhis, Pierre Adenot, Gaëtan Lehmann, Christophe Audouard, Jean-Paul Renard, Xavier Vignon, and Nathalie Beaujean

Efficient reprograming of the donor cell genome in nuclear transfer (NT) embryos is linked to the ability of the embryos to sustain full-term development. As the nuclear architecture has recently emerged as a key factor in the regulation of gene expression, we questioned whether early bovine embryos obtained from transfer of cultured fibroblasts into enucleated oocytes would adopt an embryo-like nuclear organization. We studied the dynamics of constitutive heterochromatin in the stages prior to embryonic genome activation by distribution analysis of heterochromatin protein CBX1 (HP1), centromeric proteins CENPA and CENPB, and histone H3 three-methylated at lysine 9. Then we applied descriptive, quantitative, and co-localization analyses. A dramatic reorganization of heterochromatic blocks of somatic donor cells was first observed in the late one-cell stage NT embryos. Then at two- and four-cell stages, we found two types of NT embryos: one displaying noncondensed heterochromatin patches similar to IVF embryos, whereas the second type displayed condensed heterochromatin blocks, normally observed in IVF embryos only after the eight-cell stage. These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei. The relationship with the somatic nucleus reprograming efficiency is discussed.

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Cai-Xia Yang, Zichuan Liu, Renaud Fleurot, Pierre Adenot, Véronique Duranthon, Xavier Vignon, Qi Zhou, Jean-Paul Renard, and Nathalie Beaujean

To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1β and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.