Nucleolar Material Research Articles

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera)

Aspects of nucleolar activity during spermatogenesis were assessed in three triatomine species ( Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans) using cytochemical and fluorescent staining techniques. Toluidine blue and a variant of critical electrolytic concentration (CEC) allowed the discrimination of rRNA providing structural details of the nucleolus and RNA distribution during meiotic cell division. Acridine orange fluorochrome staining permitted the differentiation of nucleic acids, and silver-ion impregnation made possible the observation of pre-nucleolar bodies (PNBs). Our results support the phenomenon known as “persistence of the nucleolar material”, and the hypothesis of post-meiotic reactivation of rRNA genes. Nucleolar organizer regions (NORs) were observed in some metaphasic spermatogonial chromosomes in P. megistus and T. infestans. In P. megistus at diplotene–diakinesis, NORs were also detected in one of the sex chromosomes and in an autosome. Therefore, it may be inferred that, in triatomines, the nucleolus does not completely disappear, but persists in the form of small bodies that get together to form the next nucleolar cycle which, in the case of meiosis, will be completed if fertilization occurs and a new zygote is formed.

Meiotic nucleolar cycle and chromatoid body formation during the rat ( Rattus novergicus) and mouse ( Mus musculus) spermiogenesis

The aims of the present study were to follow the nucleolar cycle in spermiogenesis of the laboratory rodents Rattus novergicus and Mus musculus, to verify the relationship between the nucleolar component and chromatoid body (CB) formation and to investigate the function of this cytoplasmic supramolecular structure in spermatogenic haploid cells. Histological sections of adult seminiferous tubules were analyzed cytochemically by light microscopy and ultrastructural procedures by transmission electron microscopy. The results reveal that in early spermatids, the CB was visualized in association with the Golgi cisterns indicating that this structure may participate in the acrosome formation process. In late spermatids, the CB was observed near the axonema, a fact suggesting that this structure may support the formation of the spermatozoon tail. In conclusion, our data showed that there is disintegration of spermatid nucleoli at the beginning of spermatogenesis and a fraction of this nucleolar material migrates to the cytoplasm, where a specific structure is formed, known as the “chromatoid body”, which, apparently, participates in some parts of the rodent spermiogenesis process.

Amitosis, including nucleolar behaviour during fragmentation, in both axial and corticating cells of Chara contraria (Charales, Charophyta)

A.A. Vouilloud, E.J. Cáceres and P.I. Leonardi. 2007. Amitosis, including nucleolar behaviour during fragmentation, in both axial and corticating cells of Chara contraria (Charales, Charophyta). Phycologia 46: 178–186. DOI: 10.2216/06-38.1The results from the present study indicate that in Chara contraria the morphology of amitotic resting nuclei and amitosis depend essentially on the cell type as well to on some extent on the age and development of the cells. Young axial cells of principal axes and branches exhibited C-shaped resting nuclei. During amitosis nuclei initially stretched out and became helical shaped, and they then divided themselves into two similar C-shaped nuclei that remained close to each other. The nucleolar material gradually merged together to eventually form a central nucleolus, which adopted the curving of the nucleus. Spindle-shaped resting nuclei with numerous and irregularly distributed ovoid nucleolar structures were also observed. They duplicated their length during amitosis, underwent a constriction in the middle portion, and finally broke obliquely giving rise to two daughter nuclei, both with similar size. Young corticating cells exhibited initially ovoid nuclei. Then, they gradually stretched out concomitantly with the extension of the cells, giving rise to worm-shaped nuclei that bent irregularly and eventually divided themselves by constriction in portions of different sizes. The ultrastructure of amitotic nuclei in corticating cells was studied. The nuclear envelope remained intact in dividing nuclei. In section, resting nuclei exhibited numerous, small nucleolar profiles homogeneously distributed. In dividing nuclei, in contrast, few, large nucleolar profiles occupied the middle portion of the nucleus. Bundles of 3–66 tubular elements c. 20 nm in diameter ran approximately parallel to the long axis of the nucleus. Tubules were made of circular subunits c. 2 nm in diameter in cross section, and frequently they contacted nucleolar profiles and the inner membrane of the nuclear envelope.

A New Nucleolar Body Appears inDrosophila saltansSalivary Gland Cells Before Histolysis, in Programmed Cell Death

The salivary glands of Drosophila saltans (saltans group, saltans subgroup) analyzed in an advanced stage of programmed cell death showed the appearance of a single, round, nucleolar corpuscle inside the highly altered nucleus of every gland cell, at a time during which the integrity of the original nucleolus was already lost and the original nucleolar material apparently disappeared. In the same nuclei, which already had also lost the characteristic chromosome structure, some delicate chromosome threads were maintained. In many cells, the new nucleolar corpuscle and these chromosome threads are associated. These findings are novel. However, the hypothesis put forward concerning their meaning remains dependent on other studies.

Surveillance of nuclear-restricted pre-ribosomes within a subnucleolar region of Saccharomyces cerevisiae.

We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1-2 mutation, pre-60S particles were rapidly degraded following transfer to 37 degrees C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1-2 strains at 37 degrees C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1-2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.

High Resolution Mapping of Ribosomal DNA in Early Mouse Embryos by Fluorescence In Situ Hybridization1

The nucleolar precursor bodies (NPBs) are numerous discrete entities present in the nuclei of early mammalian embryos, which structurally support active rRNA genes. However, whether all rRNA genes, including those not transcribed, are spatially associated with NPBs, and moreover what is the general arrangement of ribosomal DNA (rDNA) in early mouse embryos, still remain unanswered questions. In our study, we examined the localization of rDNA in transcriptionally silent (one-cell and early two-cell) and transcriptionally active (late two-cell) mouse embryos by highly sensitive fluorescence in situ hybridization with probes complementary to mouse rDNA repeats. The results obtained showed that irrespective of the rDNA transcriptional status, one or more NPBs per nucleus were not structurally associated with rDNA. These observations support the idea that NPBs are heterogeneous in their ability to recruit rRNA genes and thus to participate in reassembly of the mature nucleolus. As in somatic cells, and despite the absence of the characteristic nucleoli, the general arrangement of rRNA genes in early mouse embryos reflected the intensity of rDNA transcription. Ribosomal RNA genes were unequally distributed with respect to repeat putative copy numbers between nucleolar organizing region (NOR)-bearing chromosomes at the first cleavage division, and more strikingly, between sister chromatid NORs of a single nucleolar organizing chromosome. The latter indicates that sister chromatids might harbor various numbers of rRNA gene copies, and that the genes might be unequally distributed between the two blastomeres during the first cleavage mitosis.

Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster

The evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sex-linked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterized by intra-species variation in hybridization signal size, appeared on the fourth chromosome related to heterochromatic rearrangements. Nucleolar material is thought to be required for sex chromosome pairing and disjunction in a variety of organisms including Drosophila. Thus, either remnant sequences, possibly intergenic spacer repeats, are still present in the sex chromosomes which have lost their NORs (as observed in D. simulans and D. sechellia), or an alternative mechanism has evolved.

Ultrastructure and geographic distribution of the genus Paradermamoeba (Gymnamoebia, Thecamoebidae)

The genus Paradermamoeba includes two species with a distinct and unusually thick cell coat. This coat consists of densely packed helical glycostyles, up to 520 nm in length. Perhaps due to the impenetrable properties of the cell coat, we were unable to achieve satisfactory quality of EM fixation in these species until recently. In this study the treatment of cells with dilute Triton X-100 detergent prior to fixation has improved preservation of the ultrastructure of both Paradermamoeba species. Paradermamoeba valamo has an unusual central nucleolus consisting of densely packed fibrils of nucleolar material. A newly isolated strain of P. levis from a UK pond, Priest Pot, forms rounded, single-walled cysts. Both species have enigmatic trichocyst-like bodies in the cytoplasm. Despite the presence of a thick and highly differentiated cell coat, dictyosomes in both species are few and are not more developed than in many other species of gymnamoebae. Neither ultrastructure nor LM morphology indicates the phylogenetic position of Paradermamoeba. It seems that the family Thecamoebidae is a heterogeneous assemblage of unrelated genera. Recent isolation of both P. valamo and P. levis in Switzerland and in the UK indicates the wide distribution of both species in Europe.

Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xyp sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques.

The mitotic and meiotic chromosomes of the beetles Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xyp, and regular chromosome segregation during anaphases I and II. The chromosomes of E. atomaria are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the Xp chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. atomaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xyp in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material.

Sequential FISH analysis with rDNA genes and Ag-NOR banding in the lady beetle Olla v-nigrum (Coleoptera: Coccinellidae).

We have characterized the meiosis of Olla v-nigrum by standard analysis, performed a NOR study using NOR banding, FISH of rDNA genes and sequential FISH/AgNOR analysis, and adapted the FISH methodology to Coccinellidae. The chromosome number determined at metaphase I was n = 9 + Xyp. At zygotene it was possible to identify the sex vesicle which presented a deeply stained heteropycnotic block. Chromosome X is much larger than the y and the two combine, forming a "parachute" in metaphase I. FISH analysis using a probe of rDNA genes 18S, 28S and 5.8S of D. melanogaster was used to map the genes in the sex vesicle. The NOR band showed high gene activity in this region. These results were confirmed using sequential FISH/Ag NOR analysis. The data obtained for Olla v-nigrum agree with the classical hypothesis raised to explain the type of sex chromosome association in a parachute format (Xyp) as being due to the presence of nucleolar material. The chromosome number and parachute configuration during metaphase I in this species agree with the basic karyotype of most Coleopterans. The major adaptation of the FISH method was the simultaneous denaturation and hybridization that permitted preservation of chromosome morphology, an essential factor when the chromosomes are small.

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: Two New Amitochondrial Heterolobosean Amoebae from Anoxic Environments

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Ultrastructural differences in bovine morulae classified as high and low qualities by morphological evaluation

Bovine morulae collected from superovulated cows were classified into four groups (excellent, good, fair, poor) using morphological evaluation under a phase contrast microscope. The number of blastomeres in morulae classified as excellent and good (high quality) was significantly higher than the number in fair and poor (low quality) morulae. The ultrastructure of morulae in each group was compared. In morulae classified as excellent and good, two characteristic cell types could be identified by the electron density of their cytoplasm and by their ultrastructural features. One type (presumptive trophoblast cells) was located in the peripheral layer of the embryo mass, was generally characterized by low electron density of cytoplasm, and possessed numerous cellular organelles such as mitochondria and Golgi apparatus and had numerous microvilli projecting into the perivitelline space. The other type (presumptive embryonic cells) was located in the interior of the embryo mass and distinguished by cytoplasm that stained more densely than that of the lighter-appearing cells. The darker-appearing cells generally possessed fewer organelles than the lighter cells, but many lysosome-like structures were present in the cytoplasm. All high quality morulae contained well-developed mature nucleoli. Many embryos classified as fair contained both types of cells, while cells in some fair and most poor quality morulae could not be distinguished by the electron density of their cytoplasm and ultrastructural features. The morulae classified as fair and poor showed less well-developed junctional complexes between cells, less well-developed apical microvilli on the blastomeres, and contained large numbers of lipid droplets, vesicles, immature mitochondria, and nucleoli with low transcriptional activity. This study demonstrates conspicuous differences in the ultrastructural features between the high quality and the low quality morulae, suggesting that these ultrastructural features may reflect the various physiological anomalies observed in previous studies.

Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli.

Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.

Microarray expression profiles in breast cancer MCF-7 cells following exposure to benzo[a]pyrene and its diol epoxide

Maintenance of genome integrity in mammalian cells may be compromised by the concomitant accumulation of DNA damage and loss of repair functions. Polycyclic aromatic hydrocarbons are classic DNA-damaging and tumor-promoting agents. Benzo[a]pyrene (B[a]P), a prototype PAH, induces several genotoxic responses, including cell cycle arrest, oxidative stress and DNA adduct formation. Exposure of MCF-7 cells to B[a]P (1−5 M) and the B[a]P diol epoxide (BPDE) (50−100 nM) induced transient S-phase arrest, which was followed by accumulation in G2 and M and 70% loss of cell viability. In cells treated with B[a]P and BPDE, we observed segregation of nucleolar material and a drastic reduction in the number of ribosomal fibrillar centers. To identify the expression profile in cells treated with B[a]P or BPDE, we analyzed the levels of expression of 1,152 selected genes using a GeneMAP CancerArray in control and treated cells. Changes in gene expression were evaluated twice in duplicate (n=4). Compared with the expression levels in untreated cells, transcripts that were upregulated by both B[a]P and BPDE included members of the cytochrome P450, AP-1 (Fra-1, Fra-2) and glutathione S-transferase families; PARP; and G1/S-specific cyclins. In contrast, transcript levels for BRCA-1, G2/M cyclins, bcl-2, junB and fosB were downregulated in cells treated with B[a]P or BPDE. Treatment with the metabolite BPDE tended to elicit a stronger response than treatment with B[a]P. Analysis of expression profiles by microarray may be useful to understand the interactions among overlapping pathways and assess exposure to environmental carcinogens.

Characteristics of Actinomycin D-induced segregation of a "noncanonical" nucleolus of the sea urchin Paracentrotus lividus

The structure of a "noncanonical" nucleolus of vitellogenic oocytes in the sea urchin Paracentrotus lividus was studied using the inhibitor of transcription actinomycin D. In the control cells, the nucleolus consists of two separated structural subdomains: the dense fibrillar-granular peripheral area and the fibrillar central area. The nucleolus did not contain subdomains corresponding to the fibrillar center and dense fibrillar component of "typical" nucleoli. After treatment with actinomycin D, numerous argyrophilic granules appeared in the karyoplasm, the intranucleolar DNA became compact, and the nucleolar material was segregated into two or three separated zones, the residual peripheral area being the densest and largest. Lesser zones had a decreased electron density and contained argyrophilic proteins and, apparently, the nucleolar organizer material. These results suggest that, for normal rRNA expression and processing, the presence of structural subdomains in the nucleolus, such as fibrillar complexes and a dense fibrillar component, is not essential.

Effects of Al 3+ on root growth, cell division, and nucleoli in root tip cells of Zea mays L.

The effects of different concentrations (10–5–10–1 M) of aluminum chloride on root growth, cell division, and nucleoli in root tip cells of Zea mays L. were investigated. The results showed that Al inhibited root growth of Zea mays only at 10–2–10–1 M.Al3+ has a marked effect on c-mitosis. Al, at higher concentration, has toxic effects on root tip cells, causing aberration of chromosomal morphology and structure: c-mitosis, anaphase bridges, and chromosome stickiness. Al induces the appearance of some silver-stained particulate material scattered in the nuclei, and extrusion of the nucleolar material from the nucleus into the cytoplasm. Once the nucleolus was affected, and silver-stained nucleolar material was extruded from the nucleus into the cytoplasm, root growth was almost or completely stopped. The possible mechanism of the Al poisoning of root tip cells of Zea mays is also briefly discussed.

Functional Conservation and Cell Cycle Localization of the Nhp2 Core Component of H+ACA snoRNPs in Fission and Budding Yeasts

We report the identification of a novel nucleolar protein from fission yeast, p17nhp2, which is homologous to the recently identified Nhp2p core component of H+ACA snoRNPs in Saccharomyces cerevisiae. We show that the fission yeast p17nhp2 localizes to the nucleolus in live S. cerevisiae or Schizosaccharomyces pombe cells and is functionally conserved since the fission yeast gene can complement a deletion of the NHP2 gene in budding yeast. Analysis of p17nhp2 during the mitotic cell cycles of living fission and budding yeast cells shows that this protein, and by implication H+ACA snoRNPs, remains localized with nucleolar material during mitosis, although the gross organization of partitioning of p17nhp2 during anaphase is different in a comparison of the two yeasts. During anaphase in S. pombe p17nhp2 trails segregating chromatin, while in S. cerevisiae the protein segregates alongside bulk chromatin. The pattern of segregation comparing haploid and diploid S. cerevisiae cells suggests that p17nhp2 is closely associated with the rDNA during nuclear division.

The role of rDNA genes in X chromosome association in the aphid Acyrthosiphon pisum.

Silver staining of mitotic metaphases of the aphid A. pisum reveals the presence of argentophilic bridges connecting the two X chromosomes. The presence of nucleolar material connecting sex chromosomes seems to be quite a common phenomenon in organisms belonging to very different phyla, and suggests a role of nucleolar proteins in chromosome association and disjunction. In somatic cells of A. pisum, bridges connecting X chromosomes are detectable not only after silver staining but also after CMA3 staining. This finding suggests that GC rich DNA is involved in this type of association. Molecular analysis of rDNA intergenic spacers shows several 247 bp repeats containing short sequences having a high level of homology with the chi sequence of Escherichia coli and with the consensus core region of human hypervariable minisatellites. Moreover, each 247 bp repeat presents a perfect copy of a promoter sequence for polymerase I. These aphid repeats show structural homologies with a 240 bp repeat, which is considered to be responsible for sex chromosome pairing in Drosophila, not only in view of their common presence within rDNA spacers but also for their length and structure. The presence of chi sequences in the IGS of A. pisum, by promoting unequal crossing-over between rDNA genes, could thus give rise to the nucleolar organizing region (NOR) heteromorphism described in different aphid species. Although X pairing at NORs is fundamental in aphid male determination, the presence of heteromorphism of rDNA genes does not inhibit male determination in the A. pisum clone utilized for our experiments.

Ribosomal RNA transcriptional activation and processing in hamster facial motoneurons: Effects of axotomy with or without exposure to testosterone

A key step in the ability of neurons to survive injury and successfully regenerate involves ribosomal RNA production. Testosterone propionate (TP), augments facial nerve regeneration in the adult hamster. TP modulates the nucleolar reaction in injured facial motoneurons, such that mature ribosome levels increase more rapidly and in greater magnitude than with injury only. In this study, molecular and electron microscopic stereologic approaches were used to determine the effects of axotomy and steroid treatment on ribosomal transcription and processing in facial motoneurons. Castrated adult male hamsters were subjected to right facial nerve transection at the stylomastoid foramen. Half the animals were subcutaneously implanted with one Silastic TP capsule, with the remainder sham implanted. For the in situ hybridization experiments, postoperative survival times were 0.5, 2, or 6 hours. In situ hybridization with a ribosomal DNA probe specific to the external transcribed spacer region located at the 5' end of the ribosomal gene was accomplished. Transcriptional activation of the rRNA gene occurred rapidly, within 2 hours, after injury only. Unexpectedly, TP treatment did not alter the time course or magnitude of rRNA transcriptional activity. For the electron microscope experiments, the postoperative time of 12 hours was selected. Stereologic analysis of 3 nucleolar subcomponents, fibrillar centers (site of rRNA transcription), nucleolonema (site of rRNA processing), and granular material (site of preribosome storage), was accomplished. TP decreased the nucleolonemal strands and the granular material, relative to injury only. These results suggest that, although rRNA transcription is rapidly activated by axotomy, rRNA processing is temporarily stalled. TP does not affect the early, axotomy-induced transcriptional activation of the ribosomal gene, but may, instead, prevent the subsequent disruption in rRNA processing. An hypothesis for the molecular mechanism by which steroids augment the regenerative capabilities of injured facial motoneurons is presented.

Nuclear RNA is extruded from apoptotic cells.

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-containing aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.