title: High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.
abstract: Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation. Electronic address: neznanka_no@mail.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, 117997 Moscow, Russian Federation. Electronic address: khodarovich@mail.ru.
authors.forename: Yuriy M
authors.initials: YM
authors.lastname: Khodarovich
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, GSP-1, Leninskiye Gory, MSU, 1-73, Office 433, 119991 Moscow, Russian Federation. Electronic address: Lavrenteva-H@yandex.ru.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Lavrentyeva
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation. Electronic address: zatsepina_olga@mail.ru.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Zatsepina
mesh_terms: D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D020459:Genes, rRNA / Q000235:genetics*; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D012322:RNA Precursors / Q000648:ultrastructure*; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D012322:RNA Precursors; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins
keywords: BrUTP; Mouse GV oocytes; Nucleolar proteins; Nucleolus-like bodies; Pre-rRNA; Transcription; rRNA