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Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis

Published online by Cambridge University Press:  26 September 2008

Jorge M. Cuadros-Fernández  and
Pedro Esponda
Jorge M. Cuadros-Fernández
Affiliation:
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Pedro Esponda*
Affiliation:
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
*
Pedro Esponda, Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain. Telephone: +341-5611800. Fax: +341-5627518.
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Summary

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Keywords

EmbryogenesisFibrillarinMicroinjectionNucleolusRNA Pol I
Type
Article
Information
Zygote , Volume 4 , Issue 1 , February 1996 , pp. 49 - 58
Copyright
Copyright © Cambridge University Press 1996

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References

Aris, J.P. & Blobel, G. (1991). cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc. Natl. Acad. Sci. USA 88, 931–5.CrossRefGoogle ScholarPubMed
Azum-Gélade, M.-C., Noaillac-Depeyre, J., Caizergues-Ferrer, M. & Gas, N. (1994). Cell cycle redistribution of U3 snRNA and fibrillarin: presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase. J. Cell Sci. 107, 463–75.CrossRefGoogle ScholarPubMed
Baran, V., Vesela, J., Rehak, P., Koppel, J. & Fléchon, J.E. (1995). Localization of fibrillarin and nucleolin in nucleoli of mouse preimplanatation embryos. Mol. Reprod. Dev. 40, 305–10.CrossRefGoogle Scholar
Baserga, S.J., Yang, X.W. & Steitz, J.A. (1991). An intact Box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. EMBO J. 10, 2645–51.CrossRefGoogle Scholar
Benavente, R., Rose, K.M., Reimer, G., Hügle-Dörr, B & Scheer, U. (1987) Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells. J. cell. Biol. 105, 1483–91.CrossRefGoogle ScholarPubMed
Benavente, R.,Reimer, G., Rose, K.M., Hügle-Dörr, B. & Scheer, U. (1988a) Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells. Chromosoma 97, 115–23.CrossRefGoogle ScholarPubMed
Benavente, R., Schmidt-Zachmann, M.S., Hügle-Dörr, B., Reimer, G., Rose, K.M., & Scheer, U. (1988b) Identification and definition of nucleolus-related fibrillar bodies in micronucleated cells. Exp. Cell Res. 178, 518–23.Google Scholar
Biggiogera, M., Bürki, K., Kaufmann, S.H., Shaper, J.H., Gas, N., Amalric, F. & Fakan, S. (1990). Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy. Development 110, 1263–70.CrossRefGoogle ScholarPubMed
Camous, S., Kopečná, V. &Fléchon, J.-E. (1986). Autoradiographic detection of the earliest stage of the [3]-uridine incorporation into the cow embryo. Biol. Cell 58, 195–200.CrossRefGoogle ScholarPubMed
Christensen, M.E.,Beyer, A.L.,Walker, B. &Lestourgeon, W.M. (1977). Identification of N G, N G-dimethylarginine in a nuclear protein from the lower eukaryote Phyasrum Polycephalum homologous to the major Protein of mammalian 40S ribonucleoprotein particles. Biochem. Biophys. Res. Commun. 74, 621–9.CrossRefGoogle Scholar
De la Torre, C. & Giménez-Martín, G. (1982). The nucleolar cycle. In The Nucleolus, ed. Jordan, E.G & Cullis, C.A.. 153–77. Cambridge: Cambridge University Press.Google Scholar
Erard, M.S., Belenguer, P., Caizergues-Ferrer, M., Pantaloni, A. & Amalric, F. (1988). A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1. Eur. J. Biochem. 175, 525–30.CrossRefGoogle Scholar
Escande-Geraud, M.L., Azum, M.C., Tichadou, J.L. & Gas, N. (1985). Correlation between rDNA transcription and distribution of a 100 kD nucleolar protein in CHO cells. Exp. Cell res. 161, 353–63.CrossRefGoogle ScholarPubMed
Ferreira, J. & Carmo-Fonseca, M. (1995). The biogenesis of the coiled body during early mouse development. Development 121, 601–12.CrossRefGoogle ScholarPubMed
Geuskens, M. & Alexandre, H. (1984). Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos. Cell Differ. 14, 125–34.CrossRefGoogle ScholarPubMed
Girard, J.-P., Feliu, J., Caizergues-Ferrer, M. & Lapeyre, B. (1993). Study of multiple fibrillarin mRNAs reveals that 3′ end formation in Schizosaccharomyces pombe is sensitive to cold shock. Nucleic Acids Res. 21, 1881–7.Google Scholar
Goessens, G. (1984). Nucleolar structure. Int. Rev. Cytol. 87, 107–58.CrossRefGoogle ScholarPubMed
Hillman, N. & Tasca, R.J. (1969). Ultrastructural and autoradiographic studies of mouse cleavage stages. Am. J. Anat 126, 151–74.CrossRefGoogle ScholarPubMed
Hughes, J.M.X. & Ares, M. Jr. (1991). Depletion of U3 small nucleolar RNA inhibits cleavage in the 5′ external transccribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J. 10, 4231–9.CrossRefGoogle ScholarPubMed
Jansen, R. P., Hurt, E.C., Kern, H., Lehtonen, H., Carmo Fonseca, M., Lapeyre, B. &Tollervey, D. (1991). Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast. J. Cell Biol. 113, 715–29.CrossRefGoogle ScholarPubMed
Jiménez-García, L.F., Segura Valdez, M.L., Ochs, R.L., Rothblum, L.I., Hannan, R. & Spector, D.L. (1994). Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis. Mol. Biol. Cell 5, 955–66.CrossRefGoogle ScholarPubMed
Jordan, E.G. (1984). Nucleolar nomenclature. J. Cell Sci. 67, 217–20.CrossRefGoogle ScholarPubMed
Kass, S., Tyc, K., Steitz, J.A. & Sollner-Webb, B. (1990). The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell 60, 897–908.CrossRefGoogle ScholarPubMed
King, W.A., Chartrain, I., Kopečný, V., Betteridge, K.J. & Bergeron, H. (1989). Nucleolus organizer regions and nucleoli in mammalian embryos. J. Reprod. Fertil. Suppl. 38, 63–71.Google ScholarPubMed
Kleijmeer, M.J., Kelly, A., Geuze, H.J., Slot, J.W., Towsnsend, A. & Trowsdale, J. (1992). Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi. Nature 357, 3424.CrossRefGoogle ScholarPubMed
Kopečný, V., Fléchon, J.-E., Camous, S. & JrFulka, J. (1989). Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine-structural autoradiographic study. Mol. Reprod. Dev. 1, 79–90.CrossRefGoogle ScholarPubMed
Kopečný, V., Fakan, S., Pavlok, A., Pivko, J., Grafenau, p., Biggiogera, M., Leser, G. & Martin, T.E. (1991). Immunoelectron microscopic localization of small nuclear ribonucleoproteins during bovine early embryogenesis. Mol. Reprod. Dev. 29, 209–19.CrossRefGoogle ScholarPubMed
Martin, M. & Medina, F.J. (1991). A Drosophila anti-RNA Polymerase II antibody recognizes a plant nucleolar antigen, RNA polymerase I, which is mostly loclizedin fibrillar centres. J. cell Sci. 100, 99–107.CrossRefGoogle ScholarPubMed
Newman, G.R., Jasani, B. & Williams, E.D. (1983). A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochem. J. 15, 543–55.CrossRefGoogle ScholarPubMed
Ochs, R.L. & Smetana, K., (1991). Detection of fibrillarin in nucleolar remnants and the nucleolar matrix. Exp. Cell Res. 197, 183–;90.Google Scholar
Ochs, R.L., Lischwe, M.A., Shen, E., Carroll, R.E. & Busch, H. (1985 a). Nucleologenesis: composition and fate of Prenucleolar bodies Chromosoma 92, 330–6.CrossRefGoogle ScholarPubMed
Ochs, R.L., Lischwe, M.A., Spohn, W.H. & Busch, H. (1985 b). Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol. Cell 54, 123–4.CrossRefGoogle ScholarPubMed
Parker, K.A. & Steitz, J.A. (1987). Structural analyses of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-RNA. Mol. Cell Biol. 7, 2899–913.Google Scholar
Pavlok, A., Kopenčný, V., Lucas-Hahn, A. &Niemann, H. (1993). Transcriptional activity and nuclear ultrastructure of 8-cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles. Mol. Reprod. Dev. 35, 233–43.CrossRefGoogle ScholarPubMed
Pinto-Correia, C., Long, C.R., Chang, T. & Robl, J.M. (1995). Factors involved in nuclear reprogramming during early development in the rabbit. Mol. Reprod. Dev. 40, 292–304.CrossRefGoogle ScholarPubMed
Prather, R., Simerly, C., Schatten, G., Pilch, D.R., Lobo, S.M., Marzluff, W.F., Dean, W.L. & Schultz, G.A. (1990). U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs. Dev. Biol. 138, 247–55.CrossRefGoogle Scholar
Rose, K.M., Stetler, D.A. & Jacob, S.T. (1981). Protein kinase activity of RNA polymerase I purified from a rat hepatoma: Probable function of Mr 42000 and 24600 Polypeptides. Proc. Natl. Acad. Sci. USA 78, 2833–7.CrossRefGoogle Scholar
Rothblum, L.I., Mamrack, P.M., Kunkle, H.M., Olson, M.O.J & Busch, H. (1977) Fractionation of nucleoli: enzymatic and two-dimensional polyacrylamide gel electrophoretic analysis. Biochemistry. 16, 4716–21.Google Scholar
Scheer, U. & Benavente, R. (1990). Functional and dynamic aspects of the mammalian nucleolus. BioEssays. 12, 14–21.CrossRefGoogle ScholarPubMed
Scheer, U. & Rose, K.M. (1984). Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc. Natl. Acad. Sci. USA 81. 1431–5.CrossRefGoogle ScholarPubMed
Scheer, U. & Weisenberger, D. (1994). The nucleolus. Curr. Opin. Cell Biol. 6, 354–9.CrossRefGoogle ScholarPubMed
Schimmang, T., Tollervey, D., Kern, H. & Hurt, E.C. (1989). A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 8, 4015–24.CrossRefGoogle ScholarPubMed
Spector, D.L., Ochs, R.L. & Busch, H. (1984). Silver staining, immunofluorescence and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23. Chromosoma. 90, 139–48.CrossRefGoogle ScholarPubMed
Takeuchi, I.K. & Takeuchi, Y.K. (1986). Ultrastructural localization of Ag-NOR proteins in full-grown oocytes and preimplantation embryos of mice. J. Electron Microsc. 35, 280–7.Google ScholarPubMed
Terns, M.P. & Dahlberg, J.E. (1994). Retention and 5' cap trimethylation of U3 snRNA in the nucleus. Science 264, 959–61.CrossRefGoogle ScholarPubMed
Tesařík, J., Kopečný, V., Mandelbaum, J., Da large, C. & Fléchon, J.-E. (1986). Nucleologenesis in the human embryo developing in vitro: ultrastructural and autoradiographic analysis. Dev. Biol. 115, 193–203.CrossRefGoogle ScholarPubMed
Tesařík, J., Kopečný, V., Plachot, M. & Mandelbaum, J. (1987). High-resolution autoradiographic localization of DNA-containing sites and RNA synthesis in developing nucleoli of human preimplantation embryos: a new concept of embryonic nucleologenesis. Development 101, 777–91.CrossRefGoogle ScholarPubMed
Tollervey, D., Lehtonen, H., Carmo-Fonseca, M. & Hurt, E.C. (1991). The small nucleolar RNP protein NOP1 (fibrillarin)is required for pre-rRNA processing in yeast. EMBO J. 10, 573–83.CrossRefGoogle ScholarPubMed
Tollervey, D., Lehtonen, H., Jansen, R., Kern, H. & Hurt, E.C. (1993). Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell 72, 443–57.CrossRefGoogle ScholarPubMed
Weisenberger, D. & Scheer, U. (1995). A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis. J. Cell Biol. 129, 561–75.CrossRefGoogle ScholarPubMed