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Summary In mouse oocyte during meiotic prophase I, rRNA synthesis increases from midpachytene (onset of nucleologenesis) to the diplotene stage (both 4C DNA containing). During these stages the nucleoli undergone morphological changes from the ring-shaped structure to the nucleolonemal type. The results we obtained by 3-D reconstruction after serial sectionning of different nuclei during these stages, showed that there is an increase in the number of fibrillar centers (from 5–7 to 97–113) in correlation with the development of the nucleolonema. This result demonstrated that the fibrillar centers cannot be considered only as the interphasic counterparts of the nucleolar organizers in reticulated nucleoli.

Selective silver staining demonstrated that autosomal bivalents containing transcriptively active nucleolar organizers associated with the sex vesicle during pachytene of mouse spermatocytes. Later in pachytene, the nucleolar organizers covered the portion of the sex vesicle furthest from the attachment to the nuclear envelope. Hybridization in situ revealed the presence of rDNA in the silver-positive material. The nucleolus, formed from an autosomal bivalent, exhibited a large fibrillar center surrounded by an electron-opaque fibrillar zone. The nucleolar association with the sex vesicle was studied at early, middle, and late pachytene by hybridization in situ, NOR silver staining, and electron microscopy. These observations enabled us to further define the relationships of the nucleolar components with the X-Y pair.

Classical electron-microscopic techniques (enzymic digestion, EDTA regressive staining) allied with autoradiographic studies after [3H]uridine incorporation or after RNA synthesis initiated by an exogeneous RNA polymerase in the presence of tritiated GTP, enabled us to describe the fine structure and activity of the nucleolus in an established Drosophila cell line. This nucleolus is composed of a large central multilobed core containing proteins, RNA molecules and a DNA-containing component. This core is surrounded by and connected to large clumps of dense fibrillar nucleolus-associated chromatin, which are intermingled with fibrillogranular ramifications extending from the core towards the nuclear envelope. These ramifications are covered by granules of ribosomal ribonucleoprotein. As shown by EDTA regressive staining the nucleolar core contains a ribonucleoprotein network, which unravels and ramifies within a fibrous matrix. RNA synthesis takes place at the level of this network in the internal part of the core. The molecules synthesized are associated with proteins and are exported out of the core in the form of granules. Although it is composed of the same constituents as other nucleoli, the nucleolus of Drosophila cells seems to be less organized, in that it never displays fibrillar centres, which have been referred to as the nucleolar counterparts of the nucleolus-organizers in a wide variety of organisms.

In mouse testis, the diploid Sertoli cell displays one large nucleolus flanked symmetrically by two heterochromatic masses. The hybridization in situ with [3H]rRNA confirmed that the ribosomal cistrons are localized within the central nucleolar mass. At the ultrastructural level this nucleolar mass appears to be reticulated and contains numerous fibrillar centres. These fibrillar centres are surrounded and interconnected by an electron-opaque fibrillar network, which constitutes the reticulated nucleolonema of the nucleolus. Ag-NOR staining reveals the presence of the argyrophilic proteins associated with active nucleolus-organizing regions (NORs) within both the fibrillar centres and the electron-opaque fibrillar component. Autoradiographic studies after [‘H]uridine incorporation show that ribosomal DNA transcription only takes place in this dense fibrillar component. Three-dimensional reconstruction of four Sertoli cell nucleoli after serial sectioning reveals that the size and number of the fibrillar centres are very variable from one cell to another (26, 35, 38 and 41 fibrillar centres). The analysis of the volume occupied by the fibrillar centres as compared to the whole nucleolar volume demonstrates that the larger the nucleolus, the more fibrillar centres it contains, but also the more numerous the fibrillar centres, the larger their total volume. While in each case the number of the NORs is virtually the same, i.e. ten. In the light of these results we concluded that, at least in reticulated nucleoli, there is no numerical relationship between the number of fibrillar centres and the number of NORs, and that the fibrillar centres cannot be considered only as the nucleolar counterparts of the NORs. Moreover, the increasing number of fibrillar centres from the smallest nucleolus to the largest one is difficult to explain by the previously postulated hypothesis of a reserve of inactive rDNA packaged in the fibrillar centres. These data led us to reconsider the role of the fibrillar centres in the transcriptional activity of reticulated nucleoli.

In embryonic cell-line derivative KCo of Drosophila melanogaster, the nucleolus, like most nucleoli, contains a small proportion of ribosomal DNA (1–2% of the total nucleolar DNA). The ribosomal DNA is virtually the only active gene set in the nucleolus and is found among long stretches of inactive supercoiled heterochromatic segments. We have demonstrated by use of a Feulgen-like ammine-osmium staining procedure that, depending on the state of growth, more or less fibres of decondensed DNA emanating from the intra-nucleolar chromatin (which is in continuity with the nucleolus-associated chromatin) ramify and unravel within the central nucleolar core to be transcribed. The nucleolus expands or contracts with the variation of activity and could belong to a supramolecular matricial structure such as is shown after extraction of the nuclei. After a long period of exposure to high doses of actinomycin D, the central nucleolar core became an homogeneous fibrous structure that could be interpreted as an aggregate of protein skeletal elements. The mechanism of repression and derepression of the nucleolar chromatin could thus be explained by a mechanism involving in part a sub-nucleolar structure. We propose a schematic organization of the nucleolar chromatin in KCo cells of Drosophila and discuss it in relation with other nucleolar organizations.