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Ultrastructural autradiographic localization of the rRNA transcription sites in the quail nucleolar components using two RNA antimetabolites
... The fibrillar centres have often been interpreted as corresponding to the nucleolar counterparts of the active nucleolus-organizing regions of the chromosomes (Goessens & Lepoint, 1979). However, no transcription occurs within them but it is initiated at their periphery (Lafontaine & Lord, 1973;Goessens & Lepoint, 1974;Mirre & Knibiehler, 1981); so that, as pointed out by Wachtler, Ellinger & Schwarzacher (1980), this correspondence does not prove identity. Though active organizers could be obscured by the products of transcription and maturation (Miller & Beatty, 1969;Franke et al. 1979) in active KCo nucleoli, the fact that the Drosophila nucleolus never displays such structures, even in inactivated states, favours this hypothesis. ...
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.
... InXenopus vitellogenic oocyte, the reticular pattern of the active NOR is reminiscent of the skein-like NOR from both classical compact chromosomal nucleoli (Couve & Esponda, 1982) and the nucleolonemal-like nucleoli (Mirre & Stahl, 1981). However, in Xenopus oocytes, the NOR does not show any fibrillar electrontranslucent centre, as expected from chromosomal NOR studies (Mirre & Stahl, 1976;Mirre & Knibiehler, 1981). Apparently, the NOR consists only of a dense fibrillar core with emerging strings, known to be the site of active transcription (Miller, 1969;Miller, Miller & Beatty, 1969). ...
Silver staining at the electron microscopic level of the nucleolar organizers was carried out on Xenopus laevis oocytes at various stages of oogenesis.
The results indicate that a positive reaction takes place exclusively in the dense fibrillar component of the extrachromosomal nucleoli. This constituent undergoes morphological changes of distribution and architecture, which have been correlated with modifications of the transcriptional activity of the nucleoli. When nucleolar activity is reduced, during previtellogenesis, this constituent appears as dense homogeneous spherules well-segregated from the granular component. In contrast, when nucleolar activity is high, during vitellogenesis, it forms an heterogeneous area with an ill-delimited outline: it is organized into a fibrillar core with emerging skein-like strings. It thus seems that this constituent remains silver-stained throughout oogenesis.
These findings suggest that the method used would allow one to follow the evolution of the nucleolar organizer region (NOR) topography during oogenesis. Moreover, they point out facts that have relevance to the problem of the correlation between Ag stainability of NORs and nucleolar transcriptional activity.
Nucleolar ultrastrure was studied in fully grown human oocytes obtained from multilaminar preantral follicles and from follicles at different stages of antrum formation. Selective staining for ribonucleoproteins and 3H-uridine labeling were used in attempt to better understand the nature and functional significance of homogeneous dense nucleoli found in oocytes from large antral follicles.
There was an apparent increase in the radio of nucleonema to nucleolar interstices, accompanied by a gradual degranulation of the nucleolonema during early stages of antrum fromation. The process of nucleolar homogenization continued in oocytes from medium-size antralfollicles, island of more tightly packed fibrils being hybothesized to represent persisting active transcription units. Entirely filamentous and homogeneous nucleoli were typical for oocytes from large antral follicles. They were demonstrated to ribonucleoprotein filaments embedded in pale- staining matrix. They were demonstrated to contain newly synthesized RNA after a 30-min pulse with 3H-uridine. The described nucleolar transformations are interpreted as acorrelate of nucleolar transition from a site of RNA synthesis into a site of RNA Storage during in human oocyte preovulatory development.
The distribution of rDNA and the uptake of tritiated uridine was investigated in nucleoli of human Sertoli cells. The nucleolar components in these cells are spatially arranged in a highly ordered and invariable way and can be recognized in both light and electron microscopy. The pattern of distribution of rDNA and the pattern of uridine uptake in these nucleoli correspond to the distribution of the dense fibrillar component but cannot be correlated to the shape and size of the fibrillar centers in these cells. It therefore can be concluded that the dense fibrillar component, and not the fibrillar centers, is the site of rDNA location and transcription in nucleoli of human Sertoli cells.
This review addresses the problem of understanding nucleolar morphology in terms of nucleolar function. Nucleolar morphology and function are outlined to serve as a basis for reviewing in situ-cytochemical results that have, so far, not led to a generally accepted view on the structure-function correlation for nucleoli. Nucleoli in meiotic cells are dealth with in detail, because they illustrate the relationship between chromosomes and nucleoli particularly well. Spontaneous and induced changes in nucleolar morphology are presented and an attempt is made to explain particular morphologies in terms of functional states. The use of nucleolar morphology as a tool in diagnostics is critically evaluated.
Ultrastructural data on the third abdominal ganglion of the crayfish was heretofore only available for adult individuals. The fine structure of neurons in the adult that are involved in the escape response has been described in detail, but no similar data existed for the postnatal individual. An increase in the number of neurons in the third abdominal ganglion during postnatal stages had been reported, which suggested that several changes in the features of neurons may occur. Here we describe the general anatomy and ultrastructure of the early postnatal third abdominal ganglion, with emphasis on neurons, and we compare their characteristics to those of the adult. Abdominal ganglia of 56 crayfish of 0, 8, 10, 18, 25, 50, 110, and 150 postnatal days were processed under cacodylate buffered aldehyde fixatives, osmicated, embedded in plastic, sectioned, and examined by light and electron microscopy. The anatomy of postnatal ganglia is homologous to the anatomy of the adult ganglia except that the perineurium is not developed in postnatals. The area of neurons within the postnatal ganglion shows no stratification, but neurons are grouped in nuclei according to their size. Neurons constitute a homogeneous population in different stages of maturity, as revealed particularly by the ultrastructure of the nucleolus. Postnatal development is evident in the perineurium, which may provide structural support to the ganglion.
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.
In this study, the effects of axotomy on the ultrastructure of the nucleolus and associated organelles were examined in fetal, newborn, and early postnatal facial motoneurons of the hamster. Golden hamsters used for this study were the 14-day fetus, newborn (0 days; less than 6 hr) and 2, 4, 7, and 9 days postnatal ages, with 3 animals per group. For prenatal surgeries, pregnant hamsters were anesthetized and the facial nerves severed in the fetuses via electrocautery through the uterine wall and amniotic membrane. For postnatal surgeries, the animals were anesthetized and the right facial nerve exposed and severed at its exit from the stylomastoid foramen. At the appropriate postoperative times, the animals were reanesthetized and perfused-fixed. The facial nuclear groups were dissected and processed for routine electron microscopy. Microbody and coiled body frequencies were determined from the number of neurons containing these structures per number of neurons sampled per animal in each experimental or control group and subjected to statistical analysis. Nucleolar reactive changes that occurred during this developmental sequence fell into two major categories. The first category displayed by most injured cells consisted of an initial compacting of fibrillar material and reduction in vacuolar space. The second category appeared to represent a progression from this first stage of nucleolar reactivity into degenerative changes involving a striking segregation of nucleolar components into five distinct regions. The incidence of microbodies increased as a result of axotomy, whereas the presence of coiled bodies decreased at the later postoperative stages in the older animals. With increasing age and nucleolar maturation, the nucleolar reactive pattern became less pronounced and severe, and neuronal survival predominated. It appears, therefore, that the two categories of nucleolar changes following axotomy during early development correlate with changes observed in nucleoli under conditions of rRNA downregulation. It is hypothesized from these results that a key step in the ability of neurons to survive axotomy and successfully regenerate at these early developmental stages occurs at some point in ribosomal RNA transcription and/or processing. Complementary information at the molecular level concerning changes in nucleolar synthetic activity and ribosome production will be necessary to test this hypothesis.
In this study, progressive developmental changes in the nucleus and associated organelles, including the nucleolus, coiled bodies, nuclear envelope, and nucleoplasm, of hamster facial motor neurons were characterized by two parallel analyses: ultrastructural and morphometric. Golden hamsters (Mesocricetus auratus) used for this series were the 14-day fetus, newborn (<6 hr), and 1, 2, 3, 4, 5, 7, 9, 11, and 13 days postnatal ages, with 3 animals per group. Following anesthesia and perfusion fixation, facial nuclear groups were dissected and processed for electron microscopy. Electron micrographs and camera lucida tracings of nuclear profiles were collected and analyzed. The ultrastructural analysis revealed progressive changes in the nucleolus from a compact, segregated type to a reticulated form characteristic of actively protein-secreting cells. Nucleolar microbodies and fibrillar centers were seen at all ages; the latter structures appeared to decrease in size and increase with age in the series. The nucleolus-associated chromatin became less condensed, suggesting an increase in the incorporation of rDNA into the nucleolus proper. Coiled bodies, both free and attached to nucleoli, were found in varying frequencies. The nucleoplasm of neurons at the earliest stages contained large numbers of heterochromatin clumps, which decreased concomitantly with an increase in interchromatin granules and fibrils during the later stages. Nuclear envelope invaginations, polarized along one side of the nucleus, increased throughout the developmental period examined. These changes occurred in concert with a 61% increase in nuclear size and a 47% increase in the length of nuclear envelope. The sequence of nuclear changes observed during this early period of normal facial neuronal growth completes the study of a series of distinctly defined cytomorphic events in this cell type, the lability of which can be experimentally tested for their functional roles in neuronal development.
The distribution of ribosomal DNA (rDNA) in the nucleoli of human lymphocytes was revealed by in situ hybridization with a nonautoradiographic procedure at the electron microscopic level. rDNA is located in the dense fibrillar component of the nucleolus but not in the fibrillar centers. In the same cells the incorporation of tritiated uridine takes place in the dense fibrillar component of the nucleolus as seen by autoradiography followed by gold latensification. From these findings it can be concluded that the transcription of ribosomal DNA takes place in the dense fibrillar component of the nucleolus.
The nucleolar organizer regions (NORs) have been demonstrated to be the morphological sites around which the nucleoli develop at the end of mitosis. This chapter describes the structural and functional aspects of NORs of human chromosomes and discusses the clinical significance of these regions. The chromosomal associations resulted from NORs play an important role in at least three types of chromosomal disorders, and the most frequent of them is the meiotic nondisjunction causing trisomic condition. The degree of NOR activity is represented by different types of nucleoli, such as small ring-shaped nucleoli with low activity and compact nucleoli with full activity. The human acrocentric chromosomes because of the presence of NORs on short arms have a tendency to remain associated with each other during the cell division, which have deleterious consequences through meiotic nondisjunction. However, the biological and clinical implications of NOR size heteromorphisms of human acrocentrics are poorly understood.
A variety of cells (unstimulated human lymphocytes, phytohemagglutinin-stimulated human lymphocytes, diploid human fibroblasts, human melanoma cells, and Hela cells) were subjected in vitro to inhibition of protein biosynthesis by puromycin. Hela cells were also treated with actinomycin D to inhibit RNA-synthesis. Under puromycin treatment, the fibrillar centers of the nucleoli were smaller in all actively dividing cell types, whereas in small inactive lymphocytes from peripheral blood the inhibition of protein synthesis had no noticeable effect. Nucleoli with nucleolonema changed into compact nucleoli under puromycin treatment. When RNA-synthesis was inhibited, the fibrillar centers remained at an approximately constant volume.
These findings indicate that proteins localized in the fibrillar centers are involved in, and are used up during, rDNA-transcription and/or further steps of ribosome biogenesis. The changes in nucleolar architecture after the inhibition of protein synthesis suggest that transcriptional processes become concentrated near sites where proteins have been stored, i.e. the fibrillar centers.
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.
During meiotic prophase I the nucleolus of the mouse oocyte assumes a reticulate structure of 'nucleolonema' type. This change coincides with the appearance of several secondary fibrillar centres. The number of these centres at diplotene (97-113), largely exceeds that of nucleolar organizers (4c DNA = 20 NORs). The quantitative analysis of autoradiographs after hybridization in situ with 3H-uridine labelled rRNA, enabled us to demonstrate that the multiplication of the fibrillar centres in mouse oocyte nucleolus during meiotic prophase I is not the result of an amplification of the rDNA. The number of silver grains in pachytene and diplotene nuclei was twice that counted for somatic cell and oogonium nuclei (2c DNA).
This review attempts to document the most relevant data currently available on the in situ localization of nucleolar chromatin on plant cells. The data provided by the most powerful and recent in situ techniques, such as DNA specific ultrastructural staining, immunogold labelling, in situ molecular cytochemistry, in situ hybridization or confocal microscopy, are summarized and discussed in the light of the potential and limitations of each individual methodology. The presence of DNA in both fibrillar centres and regions of the dense fibrillar component is extensively documented. Data on the nucleolar distribution of other important macromolecules involved in ribosomal transcription are also shown and referred to with regard to the location of DNA. The comparison with the available data on the animal cell nucleolus points towards models of similar functional organization in both plant and animal nucleoli.
This article deals with the structural and functional organization of polytene chromosomes in mammals. Based on cytophotometric, autoradiographic, and electron microscopic data, the authors put forward a concept of nonclassic polytene chromosomes, with special reference to polytene chromosomes in the mammalian placenta. In cells with nonclassic polytene chromosomes, two phases of the polytene nucleus cycle are described, such as the endointerphase (S phase) and endoprophase (G phase). The authors generalize that the main feature of nonclassic polytene chromosomes is that forces binding the sister chromatids are much weaker than in the Diptera classic polytene chromosomes. This concept is confirmed by comparative studies of human, mink, and fox polytene chromosomes. The final step of the trophoblast giant cell differentiation is characterized by a transition from polyteny to polyploidy, with subsequent fragmentation of the highly polyploid nucleus into fragments of low ploidy. Similarities and dissimilarities of pathways of formation and rearrangement of nonclassic polytene chromosomes in mammals, insects, plants, and protozoans are compared. The authors discuss the significance of polyteny as one of the intrinsic conditions for performance of the fixed genetic program of trophoblast giant cell development, a program that provides for the possibility of a long coexistence between maternal and fetal allogenic organisms during pregnancy.
The lateral magnocellular nucleus of the anterior neostriatum (LMAN) in birds plays an important role in songlearning processes. Recently, it has been shown that structural changes at the cellular level in males are causally related to vocal learning. Whereas males sing, females do not. This sexual difference in behavior is also reflected in sexual differences in the neuronal structure in adult birds, with males having larger neuronal somata than do females. In the present report, the size and shape of the nucleoli were investigated to determine if sexual differences were also present at the nucleolar level. The data demonstrated a strong sexual difference in nucleolar size in both juvenile and adult birds, the cross-sectional area of the nucleoli being significantly larger in males than in females (30% and 50% larger in juvenile and adult birds, respectively). This difference between males and female finches was also reflected by the cross-sectional area of a specific type of nucleolus exhibiting a central light area. In both sexes, nucleoli exhibiting a central light area were significantly larger in juvenile and adult birds than nucleoli that lacked a central light area. The percentage of neurons exhibiting a central light area was higher in adult males than in adult females, but not in juvenile birds. The time course of development of nucleoli exhibiting a central light area in males was very similar to the development of neuronal somata size in LMAN neurons. The larger size of the nucleoli in LMAN neurons in males and the developmental changes in the incidence of nucleoli exhibiting a central light area may be indicative of a high level of ribosome production necessary for song-learning processes to occur.
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