Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA

Abstract

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.

Reconstitution of the oocyte nucleolus in mice through a single nucleolar protein, NPM2

“…2B, right). Thus, proteins are structurally important for the oocyte nucleolus and, as reported previously, RNAs are also included in the oocyte nucleolus (Shishova et al, 2015).…”

“…Moreover, the deletion of NLP in S2 cells showed similar defects to those in parthenotes/zygotes from enucleolated and Npm2-null oocytes (Liu et al, 2006;Padeken et al, 2013), and NLP binds to CTCF and the centromere-specific nucleosome CenH3, as well as to the NCL homolog Modulo, whose binding is required for centromere clustering to the nucleolus. Since NCL is one of the oocyte nucleolus proteins that was identified in our proteomics analysis as well as in other studies (Fulka and Langerova, 2014;Shishova et al, 2015), it is highly possible that NPM2 binds to NCL and is involved in centromere clustering to the nucleolus and regulation of the centromeric/pericentromeric chromatin organization in interphase, which is required for correct chromosome segregation.…”

“…Previous studies reported that some somatic-type nucleolar proteins localize at the oocyte nucleolus only when oocytes were treated with proteinase K or in response to applying an antigen-retrieval protocol (Fulka and Langerova, 2014;Shishova et al, 2015). NPM2 is the only protein whose endogenous localization at the oocyte nucleolus has been observed without any treatment (Inoue and Aoki, 2010).…”

Ribosomal RNA gene functioning in avian oogenesis

“…It is retained as the so-called nucleolus-like body (NLB), sometimes referred to as the nucleolus precursor body (NPB). The NLB contains nucleolar proteins involved in all the main steps of ribosome biogenesis but not rRNA, presumably serving as a store of nucleolar proteins (Shishova et al 2015a(Shishova et al , 2015b. NLBs have been repeatedly documented to be essential for embryonic development (Ogushi et al 2008;Fulka and Aoki 2016); however, more recent data on their possible roles seem to be quite controversial.…”

Immunolabeling of “Nucleoli” in Mouse Fully-Grown Oocytes and One-Cell Embryos is Dependent on Upstream Molecular Fixatives

“…The oocytes of both types are capable of gene expression [16]. However, the rRNA synthesis has only been described in the NSN-type oocytes [1,20,24], whereas the SN-type oocytes possess better developmental capacities [18]. The nucleolar precursor bodies (NPBs) are formed in zygotic pronuclei shortly after fertilization and are remarkably similar to NLBs of GV oocytes in morphology [6,7].…”