It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called "nucleolus-like bodies" (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA.

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Early embryos of all mammalian species contain morphologically distinct but transcriptionally silent nucleoli called the nucleolar precursor bodies (NPBs), which, unlike normal nucleoli, have been poorly studied at the biochemical level. To bridge this gap, here we examined the occurrence of RNA and proteins in early mouse embryos with two fluorochromes - an RNA-binding dye pyronin Y (PY) and the protein-binding dye fluorescein-5'-isothiocyanate (FITC). The staining patterns of zygotic NPBs were then compared with those of nucleolus-like bodies (NLBs) in fully grown surrounded nucleolus (SN)-type oocytes, which are morphologically similar to NPBs. We show that both entities contain proteins, but unlike NLBs, NPBs are significantly impoverished for RNA. Detectable amounts of RNA appear on the NPB surface only after resumption of rDNA transcription and includes pre-rRNAs and 28S rRNA as evidenced by fluorescence in situ hybridisation with specific oligonucleotide probes. Immunocytochemical assays demonstrate that zygotic NPBs contain rRNA processing factors fibrillarin, nucleophosmin and nucleolin, while UBF (the RNA polymerase I transcription factor) and ribosomal proteins RPL26 and RPS10 are not detectable. Based on the results obtained and data in the contemporary literature, we suggest a scheme of NPB assembly and maturation to normal nucleoli that assumes utilisation of maternally derived nucleolar proteins but of nascent rRNAs.