Search

After fertilization, ribosomal RNA synthesis is silenced during a period which depends on the species. Data concerning the reassembly of a functional nucleolus remain scarce. We have examined by immunocytochemistry, Western blots, and BrUTP microinjection the dynamics of major nucleolar proteins during the first cycles of mouse embryogenesis, in relation to rDNA transcription sites and coilin, a marker of Cajal bodies. We show that: (1) the reinitiation of rDNA transcription occurs at the two-cell stage, 44-45 h after hCG injection (hphCG), at the surface of the nucleolar precursor bodies (NPBs), where the RNA polymerase I (pol I) transcription complex is recruited 4-5 h before; (2) the NPBs are not equal in their ability to support recruitment of pol I and rDNA transcription; (3) maternally inherited fibrillarin undergoes a dynamic redistribution during the second cell stage, together with coilin, leading to the assembly of the Cajal body around 40 hphCG; and (4) the pol I complex is first recruited to the Cajal body before reaching its rDNA template. We also find that fibrillarin and B23 are both directly assembled around NPBs prior to ongoing pre-rRNA synthesis. Altogether, our results reveal a role of the Cajal bodies in the building of a functional nucleolus.

In mammalian preovulatory oocytes, rRNA synthesis is down-regulated until egg fertilization and zygotic genome reactivation, but the underlying regulatory mechanisms of this phenomenon are poorly characterized. We examined the molecular organization of the rRNA synthesis and processing machineries in fully grown mouse oocytes in relation to ongoing rDNA transcription and oocyte progression throughout meiosis. We show that, at the germinal vesicle stage, the two RNA polymerase I (RNA pol I) subunits, RPA116 and PAF53/RPA53, and the nucleolar upstream binding factor (UBF) remain present irrespective of ongoing rDNA transcription and colocalize in stoichiometric amounts within discrete foci at the periphery of the nucleolus-like bodies. These foci are spatially associated with the early pre-rRNA processing protein fibrillarin and in part with the pre-ribosome assembly factor B23/nucleophosmin. After germinal vesicle breakdown, the RNA pol I complex disassembles in a step-wise manner from chromosomes, while UBF remains associated with chromosomes until late prometaphase I. Dislodging of UBF, but not of RNA pol I, is impaired by the phosphatase inhibitor okadaic acid, thus strengthening the idea of a relationship between UBF dynamics and protein phosphorylation. Since neither RNA pol I, UBF, fibrillarin, nor B23 is detected at metaphase II, i.e., the normal stage of fertilization, we conclude that these nucleolar proteins are not transported to fertilized eggs by maternal chromosomes. Together, these data demonstrate an essential difference in the dynamics of the major nucleolar proteins during mitosis and meiosis., (Copyright 2000 Academic Press.)

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state., (Copyright 2000 Academic Press.)