New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.     

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.     

Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.     

Phosphoprotein pp135 is an essential component of the nucleolus organizer region (NOR)

The association of phosphoproteins pp135 and pp105 with distinct substructures of the nucleolus was studied by cytochemical and immunological methods at the light microscopic and electron microscopic level. Both phosphoproteins exhibited a very high affinity for silver and Giemsa staining compared to other nucleolar proteins. Immunolocalization of pp135 and pp105 during mitosis by light microscopy revealed a tight association of pp135 with the silver staining nucleolus organizer region (NOR), whereas pp105 (cross-reacting with C23) appeared to be only partially associated with the NOR, exclusively at telophase. At the immunoelectron microscopic level the distribution of pp135 and pp105 was investigated in interphase nucleoli. Phosphoprotein pp135 was located in the fibrillar shell and pp105 in the fibrillar shell and the granular zone. The fibrillar centers were essentially free of both phosphoproteins..     

Ultrastructural Distribution of DNA within Plant Meristematic Cell Nucleoli during Activation and the Subsequent Inactivation by a Cold Stress

We have investigated the precise location of DNA within the meristematic cell nucleolus ofZea maysroot cells andPisum sativumcotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with thein situterminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.     

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.     

Human ribosomal RNA gene repeats are localized in the dense fibrillar component of nucleoli: light and electron microscopic in situ hybridization in human Sertoli cells.

The distribution of the human ribosomal gene repeat within human Sertoli cell nucleoli was investigated with the help of DNA-DNA in situ hybridization at the light and electron microscopic level. Probes from both the transcribed part of the gene repeat and the "non-transcribed" spacer were found to hybridize predominantly to the dense fibrillar component of nucleoli. It therefore can be concluded that the dense fibrillar component of nucleoli is the major site of the intranucleolar location of the ribosomal DNA. This holds true not only for the dense fibrillar component adjacent to fibrillar centers, but also for the dense fibrillar component remote from the fibrillar centers.     

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.     

3-dimensional organization of the nucleolus and the nucleolus-organizing regions in differentiated cells. I. Ring-shaped nucleoli in the endotheliocytes, smooth-muscle cells and fibroblasts in the mouse

The method of ultra-thin serial sections was used to study the three-dimensional structure and to perform the quantitative analysis of ring-shaped nucleoli of kidney and liver endotheliocytes, smooth muscle cells of kidney arterioles and fibroblasts of mice. Spatial models of ring-shaped nucleoli with one fibrillar centre are given. For the quantitative analysis the following parameters were measured: the number and volumes of nucleoli, fibrillar centers, RNP-containing structures, the vacuolar system and the RNP-index (the latter is a ratio of RNP-part and fibrillar center volumes). Nucleoli of the same type of cells, occasionally in the same nucleus, were found to differ sharply in their fibrillar center shape. Differences in the mean volume values of nucleoli, fibrillar centers and the RNP-part between some cell populations are sufficiently well pronounced. Within the same population ring-shaped nucleoli have, as a rule, specific volume values of nucleoli, RNP and fibrillar centers. The comparison of quantitative data obtained on different cell types showed that the mean RNP-index values were the most stable parameter. The structural relation between fibrillar centers, intra- and perinucleolar chromatin and lacunar region is shown. The structural organization of intranucleolar chromatin and rRNA in the nucleolar body and in fibrillar centers is discussed.