Analysis of the localization of fibrillarin and sites of pre-rRNA synthesis in the nucleolus-like bodies of mouse GV oocytes after mild treatment with proteinase K

Analysis of the localization of fibrillarin and sites of pre-rRNA synthesis in the nucleolus-like... Postnatal development of mammalian oocytes is accompanied by functional and structural remodeling of the nucleolar apparatus: the final stage of this process is the formation of large objects (up to 10 μm in diameter) termed nucleolus-like bodies (NLBs) in preovulatory GV oocytes. NLB material was shown to be essential for early embryonic development, but its composition is still uncharacterized. In the present study, the protein-binding dye fluorescein-5-isothiocyanate (FITC) was used to show that proteins characterized by a high local concentration are essential NLB components in mouse GV oocytes. One of these proteins was able to be identified for the first time using a mild treatment of oocytes with proteinase K; the protein identified was fibrillarin, a factor of early pre-rRNA processing. Fibrillarin is present in the inner NLB mass of all oocytes capable of synthesizing rRNA; however, it is not colocalized with BrUTP microinjected into oocytes in order to identify transcribed ribosomal genes, in contrast to the “surface” fibrillarin. These observations imply the accumulation of nucleolar proteins not involved in ribosome biogenesis inside the NLB. All NLBs present in an individual nucleus of an NSN-type GV oocyte contain fibrillarin and are associated with active ribosomal genes. The results obtained in the present work demonstrate that proteinase K treatment of GV mouse oocytes allows for: (1) identification of “cryptic” proteins inside the densely packed NLB material and (2) the enhancement of oocyte image quality during BrUTP-based identification of rRNA synthesis sites but (3) not for the detection of active ribosomal genes in the inner mass of the NLB. The fluorescent dye FITC can be recommended for assessment of intracellular protein localization in the oocytes of all mammalian species. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Russian Journal of Developmental Biology Springer Journals

Analysis of the localization of fibrillarin and sites of pre-rRNA synthesis in the nucleolus-like bodies of mouse GV oocytes after mild treatment with proteinase K

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Publisher
Pleiades Publishing
Copyright
Copyright © 2015 by Pleiades Publishing, Inc.
Subject
Life Sciences; Developmental Biology; Animal Anatomy / Morphology / Histology
ISSN
1062-3604
eISSN
1608-3326
D.O.I.
10.1134/S1062360415030066
Publisher site
See Article on Publisher Site

Abstract

Postnatal development of mammalian oocytes is accompanied by functional and structural remodeling of the nucleolar apparatus: the final stage of this process is the formation of large objects (up to 10 μm in diameter) termed nucleolus-like bodies (NLBs) in preovulatory GV oocytes. NLB material was shown to be essential for early embryonic development, but its composition is still uncharacterized. In the present study, the protein-binding dye fluorescein-5-isothiocyanate (FITC) was used to show that proteins characterized by a high local concentration are essential NLB components in mouse GV oocytes. One of these proteins was able to be identified for the first time using a mild treatment of oocytes with proteinase K; the protein identified was fibrillarin, a factor of early pre-rRNA processing. Fibrillarin is present in the inner NLB mass of all oocytes capable of synthesizing rRNA; however, it is not colocalized with BrUTP microinjected into oocytes in order to identify transcribed ribosomal genes, in contrast to the “surface” fibrillarin. These observations imply the accumulation of nucleolar proteins not involved in ribosome biogenesis inside the NLB. All NLBs present in an individual nucleus of an NSN-type GV oocyte contain fibrillarin and are associated with active ribosomal genes. The results obtained in the present work demonstrate that proteinase K treatment of GV mouse oocytes allows for: (1) identification of “cryptic” proteins inside the densely packed NLB material and (2) the enhancement of oocyte image quality during BrUTP-based identification of rRNA synthesis sites but (3) not for the detection of active ribosomal genes in the inner mass of the NLB. The fluorescent dye FITC can be recommended for assessment of intracellular protein localization in the oocytes of all mammalian species.

Journal

Russian Journal of Developmental BiologySpringer Journals

Published: May 26, 2015

References

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