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.     

Three-dimensional distribution of Ag-NOR proteins in rat superior cervical ganglia nucleoli according to circadian rhythm

A three-dimensional reconstruction of the distribution of Ag-NOR proteins in nucleoli of sympathetic neurons of a rat killed during the dark period of its light-dark cycle was compared with previously reported analyses on the three-dimensional distribution of fibrillar centers, the high-resolution localization of these proteins, and the morphometric results. The domain occupied by these proteins appeared to far exceed that of the fibrillar centers and included the dense fibrillar RNP component. In the present material this component in turn provided partial bridging between the units consisting of the fibrillar centers plus their surrounding dense fibrillar component.     

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.     

Locating transcribed and non-transcribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy.

Immunoelectron microscopy and in situ hybridization have been used to investigate the precise location of transcribed and non-transcribed rDNA spacer sequences. Whereas a 5'-external transcribed spacer sequence is predominantly visualized in the fibrillar centers of nucleoli, a non-transcribed spacer sequence is preferentially detected in the interstices, in close contact with the fibrillar centers and which interrupt the surrounding dense fibrillar component. Occasionally these two spacers are also observed in clumps of dense nucleolus-associated chromatin. These observations provide insights into the organization of ribosomal repeats within the nucleolus.     

Distribution of fibrillar centers and silver-stained components in the nucleolus of human Sertoli cells

The nucleolus of the human Sertoli cell consistently showed three distinct, spontaneously segregated parts: 1. one or two large, silver-positive fibrillar centers; 2. strands of dense fibrillar component continuous with the dense cords surrounding the fibrillar center. These components were also silver-positive; 3. a granular, silver-negative mass. These observations show that in the human Sertoli cell the number of fibrillar centers is far lower than the diploid number of NORs. They also suggest that the fibrillar center might contain several NORs in this cell type.     

In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA

In situ hybridization using biotinylated rDNA probes and secondary antibody coupled to gold particles was developed on ultrathin sections of Lowicryl-embedded Ehrlich tumor cells for precise localization of ribosomal RNA (rRNA) and ribosomal DNA (rDNA). For the detection of rDNA, an immunocytochemical approach involving an antibody against single-stranded DNA was used in order to determine the more efficient denaturation procedure. Using this technique, rDNA can be visualized in the fibrillar centers of nucleoli, especially in their peripheral regions at the proximity of both the dense fibrils and the nucleolar interstices as well as within the latter. rDNA was occasionally detected in some clumps of dense nucleolus-associated chromatin. Besides the presence of rRNA in the ribosome-rich cytoplasmic areas and in the dense fibrillar component and the granular component of the nucleolus, rRNA was also found in the fibrillar center areas close to the boundary region to the dense fibrillar component. These results are discussed in the light of the present knowledge on the functional organization of the nucleolus.     

Three-dimensional ultrastructure and quantitative analysis of the human Sertoli cell nucleolus

The nucleolus of the human Sertoli cell displays a spontaneous segregation of its components and has only one or 2 large fibrillar centers. The 3-dimensional reconstruction and quantitative analysis of its components was undertaken using a Quantimet 900 image analysis system in order to define the spatial relationships between the dense fibrillar component and the fibrillar center and especially to investigate whether threads of dense fibrillar component exist independently, without being linked to a fibrillar center. Our 3D reconstructions demonstrated that the dense fibrillar threads or sheets were never independent of fibrillar centers. These structures belonged to a continuous network that joined the layer of dense fibrils surrounding the fibrillar center. When the nucleolus contained 2 different-sized fibrillar centers, quantitative analysis showed that there was a proportional relationship between the volume of the dense fibrillar component and the volume of the fibrillar center. These data, compared with those previously obtained by means of autoradiographic techniques, suggest that the rDNA-containing chromatin passes through the fibrillar center and unwinds from there into the dense fibrillar component.     

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.