title: 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.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, Moscow 117997, Russian Federation. Electronic address: kseniya.shishova@inbox.ru.
authors.forename: Kseniya V
authors.initials: KV
authors.lastname: Shishova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, Moscow 117997, Russian Federation; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, GSP-1, Leninskiye Gory, MSU, 1-73, Office, Moscow 119991, Russian Federation. Electronic address: lavrenteva-h@yandex.ru.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Lavrentyeva
authors.affiliation: Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa Street, 30-387 Krakow, Poland. Electronic address: jerzy.dobrucki@uj.edu.
authors.forename: Jurek W
authors.initials: JW
authors.lastname: Dobrucki
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, Moscow 117997, Russian Federation. Electronic address: zatsepina_olga@mail.ru.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Zatsepina
mesh_terms: D000165:Acridine Orange; D000818:Animals; D002466:Cell Nucleolus / Q000737:chemistry*; D019286:Endopeptidase K; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D009841:Oligonucleotides / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism
chemical_list: C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D000090243:Nucleophosmin; C060480:Rpl26 protein, mouse; D019286:Endopeptidase K; D000165:Acridine Orange; D016650:Fluorescein-5-isothiocyanate
keywords: FITC; GV oocytes; Nucleolar proteins; Nucleolus-like bodies; Proteinase K; rRNA