No abstract

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.

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.

The pH-sensitive dual-emission fluorophore SNARF-1 coupled with a laser confocal microspectrofluorimeter was used to measure the internal pH (pHi) in different subcellular and subnuclear compartments of early mouse embryos. By this method we analysed the first cell cycle of naturally fertilised embryos in order to detect possible pHi changes correlated to cellular events, particularly the onset of replication or transcription and the first mitosis. Throughout interphase, significant differences of pHi were observed between cytoplasm and pronuclei, and, even more striking, between these compartments and nucleolus precursor bodies, whose pHi was systematically lower. We could detect significant pHi change neither during the replication phase nor at the onset of zygotic transcription, but pHi increased at the end of the one-cell stage in both cytoplasm and chromatin regions, a process that seemed specifically correlated with mitosis.