Nucleologenesis in Cloned Embryos

Whatever the cells habitually used as donors for nuclear transfer, they contain active nucleoli in interphase and are capable of rebuilding them after mitosis. The purpose of the method described here is to follow the morphology and activity of nucleoli in embryonic nuclei obtained by micromanipulation. If rRNA synthesis is transitorily repressed, it is possible to check whether the morphological involution of the nucleoli is analogous to the segregation of nucleolar components obtained artificially by treatment of cells with inhibitors of rRNA synthesis such as actinomycin D (19) or to a more physiological process such as the involution of nucleoli at the end of oocyte growth (22), which is a more or less exact reverse of the normal process of embryonic nucleologenesis. Nucleologenesis may then eventually start again.

1.3.1. Cloned Rabbit Embryos

The nuclei of 32-cell stage blastomeres contain active nucleoli. Upon transfer to enucleated oocytes (2), the nuclei no longer contain reticulated nucleoli, but rather regressing forms analogous to those of mature oocytes, which are never observed in normal zygotes. These forms contain no GC but instead only a few FCs and a reduced DFC surrounding vacuoles and/or a central NPB-like residual body (Fig. 2D). Less frequently, nucleoli were reduced to an NPB with cap. Nuclear incorporation of 5-3H-uridine could no longer be visualized by autoradiography before the end of the one-cell stage. At the 2-cell stage, the nucleoli looked like NPBs in normal zygotes or like NPBs with a cap as in normal 4-cell stage embryos (Fig. 2E). From this step, the nucleologenesis was completed almost as in control embryos, although even at the 32-cell stage, the GC did not clearly reappear (Fig. 2F).

1.3.2. Cloned Bovine Embryos

Activated or nonactivated cytoplasts were compared as recipients for nuclei of day 4 to 5 blastomeres (21). In both cases, the fully reticulated nucleoli of the transferred nuclei disappeared. However in nonactivated cytoplasts, the nucleoli regressed only to multivesiculated NPBs at the 2-cell stage and then development stopped, whereas in activated cytoplasts an almost-typical compact NPB was observed and normal development ensued, although accelerated one stage ahead for the rebuilding of active nucleoli.

During the 2- and 4-cell stages of bovine clones (19), the reticulated structure of nucleoli collapsed and looked like NPBs containing fewer and fewer vacuoles. In the best cases, a reassociation of DNA with the nucleoli was obtained at the 8-cell stage and a complete, reticulated structure was rebuilt at the 16-cell stage (a slight delay). Differences resulting from donor cell types could be observed, with somatic cells giving worse results (at least delayed de- and redifferentiation associated with lower cleaving rate) than embryonic cells. A different cytoplasmic milieu may therefore be required for reprogramming embryonic or adult nuclei (23). More generally, the cell cycle stage of the donor cells and of the cytoplasts should be equivalent.

All the examples chosen and other similar studies (24,25) indicated that rRNA synthesis is generally repressed after nucleotransfer before it is reactivated at the right time in successful cases. Early defects (delay or arrest of cleavage) may be correlated with initial incomplete gene repression and/or reactivation.

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