Nucleolar Labeling Research Articles

A 116000 M r nucleolar antigen specific for the dense fibrillar component of the nucleoli

In ATT, a human autoimmune serum, we found anti-nucleolar antibodies that recognized nucleolar antigens confined to a single nucleolar compartment, the dense fibrillar component (DFC). We localized these antigens by immunoelectron microscopy in DFC of HeLa cell nucleoli both on Lowicryl sections and cryoultrathin sections without embedding. The antigens were solubilized by incubation with 2M NaCl but not by RNase or DNase treatment. The ATT serum crossreacted with rat liver nucleoli and PtK1 cell nucleoli in which immunofluorescence labelling displayed a clumpy pattern. During mitosis, the antigens dispersed in the cytoplasm until late telophase, when they gathered in the prenucleolar bodies. In human peripheral lymphocytes, or HeLa cells treated with actinomycin D, the antigens were still present but the fluorescence intensity decreased. By immunoblotting using human nuclear extracts, the ATT serum bound to a 116,000 Mr protein at dilutions up to 1:2000. The reactivity of this band diminished with actinomycin D-treated nuclear extracts. Two minor bands were also observed at 97 and 70K (K = 10(3) Mr). Immunopurification by competition or elution demonstrated that the 116K antigens were at the origin of the nucleolar labelling. This DFC marker appeared to be different from the NOR-silver-stained proteins, which in our preparations exhibited apparent molecular weights of 105, 80 and 38-40K. In addition, these 116K antigens did not exhibit the characteristics described for DNA topoisomerase I, fibrillarin or nucleolin. We propose the 116K antigen as a new marker of the DFC of the nucleoli.

Ultrastructural localization of DNA on ultrathin sections of resin-embedded tissues by the lactoferrin-gold complex.

Lactoferrin, a DNA-binding protein, was complexed to colloidal gold and applied on ultrathin sections of resin-embedded plant tissues and bacterial cultures. Optimal results were obtained when lactoferrin was tagged to colloidal gold particles at pH 9.2. Postfixation with osmium tetroxide and embedding with Epon did not prevent the accessibility of the protein towards its corresponding binding sites. In plant nuclei, labeling was observed over the dense chromatin and to a lesser extent over the dispersed chromatin. Nucleolar labeling was preferentially located over the dense fibrillar component. Gold particles were also found to be associated with chloroplasts and mitochondria. In prokaryotic cells, a dispersed labeling was noted over the cytoplasm and, in some cases, the aggregation of few gold particles suggested the presence of packed DNA fibrils. Various control experiments confirmed the specificity of the labeling pattern obtained. Lactoferrin-gold complex appears to be a valuable probe for the intracellular demonstration of DNA molecules in double-fixed and Epon-embedded tissues.

Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.

Ultrastructural localization of DNA and RNA inAllium porrum interphase cells by means of nuclease-gold complexes

Nucleic acids have been localized in Allium porrum interphase meristematic cells by means of labelling with nuclease-gold complexes, a technique which provides high resolution and improved specificity. DNase-gold labelling was observed over dense chromatin and to a lesser extent over dispersed chromatin. Nucleolar labelling was restricted to the dense fibrillar component, very few particles being located over the fibrillar centres. Labelling by the RNase-gold complex was present over both the cytoplasm and the nucleoplasm. Cytoplasm labelling was intense over the rough endoplasmic reticulum but absent over vacuoles. In the nucleoplasm many gold particles were located at the border between the condensed and the dispersed chromatin. Nucleolar labelling was intense over the granular zones but many gold particles were also seen over the dense fibrillar component. Fibrillar centres showed, however, no labelling with the RNase-gold complex. These results are consistent with previous autoradiographic and cytochemical observations carried out on the same plant material.

Histochemical approaches for the localization of steroid hormone receptors.

Histochemical identification and visualization of steroid hormone receptors remains a goal, despite earlier success with dry- and thaw-mount autoradiography. Applications of various histochemical and immunohistochemical procedures for the identification of steroid hormone target cells and subcellular binding sites are reviewed. Results obtained with dry- and thawmount autoradiography after in vivo administration of [3H] estradiol are contrasted with those obtained by in vitro liquid emulsion autoradiography, incubation with conjugated estradiol, antibodies to estradiol, or antibodies to estradiol “receptor”. Dry- and thaw-mount autoradiography in vivo after single injection of [3H] estradiol, and in vitro after tissue slice incubation with [3H] estradiol consistently show preferential nuclear concentration of radioactivity, little in cytoplasm, and none in nucleoli. In contrast, liquid emulsion autoradiography after in vitro uterine section incubation with [3H] estradiol shows no nuclear uptake, but only labeling of cytoplasm of eosinophils. Eosinophils are also labeled with conjugated estradiol. Histochemical studies with conjugated estradiol or estradiol antibodies show variable results with preferential cytoplasmic and distinct nucleolar labeling of uterine epithelial and stromal cells; however, there is only occasional nuclear labeling, and some lack of identification of target cells, when compared to results obtained with our autoradiographic techniques. The specificity and utility of certain histochemical techniques need to be established, since some of the results are probably related to technique and do not reflect biological events. Development and use of refined histochemical techniques are needed to further contribute to the clarification of the mechanism of steroid hormone action and to provide a tool for the clinical diagnosis of steroid hormone dependent tumors.

A preliminary report on the use of immunoperoxidases to study binding of estrogens in rat uteri

This work presents the results of a preliminary study using immunoperoxidases in light and electron microscopy for the detection of estrogen binding sites in rat uteri with estradiol and a synthetic estrogen (R 2858), highly specific to estrogen receptors. The presence of estradiol (or of R 2858) was detected 90 min. after injection in the endoplasmic reticulum, in the nuclear envelope and in chromatin. Four hours after injection nucleolar labeling was noted. R 2858 was particularly useful in detecting specific binding since its use greatly reduced background binding to non-specific proteins. Furthermore the validity of the immunological reaction was confirmed by numerous control experiments. In all our experiments, nuclear labeling was always observed even at 4°C, cytoplasmic labeling at 37°C was also observed but far less marked. Although these initial results are extremely encouraging, the technical constraints imposed (in order to preserve the estrogen receptors) lead to certain cytological alterations which necessitate prudence in interpretation.

Ultrastructure and activity of the nucleolar organizer in the mouse oocyte during meiotic prophase.

The mouse oocyte is the site of nucleolar synthesis during pachytene. The chromosomes containing a nucleolar organizer are attached to the nuclear envelope by their paracentromeric heterochromatin, either alone or by taking part in the formation of a chromocentre. The nucleolus appears at the junction of the paracentromeric heterochromatin with the euchromatic portion of the bivalent. In this zone, 5.0-nm-diameter fibres, thinner than those of the rest of the chromosome (10.0 nm), extend from the lateral element of the synaptonemal complex up to the nucleolar fibrillar centre in which they penetrate. At the onset of its synthesis, the nucleolus only contains the fibrillar centre and an electron-dense fibrillar component in continuity with the latter. Growth of the nucleolus often takes place in the form of a strand whose proximal end, in contact with the fibrillar centre, is formed by preribosomal fibrils and whose distal end is at first fibrillo-granular then granular. Following brief incorporation of tritiated uridine, nucleolar labelling is active in oogonia. No ribosomal RNA-synthetic activity is revealed during leptotene and zygotene. Incorporation resumes at mid-pachytene, with labelling located over the electron-dense fibrillar component adjacent to the fibrillar centre. These observations suggest that the rDNA is located in both the fibrillar centre and its associated electron-dense fibrillar component and that the rDNA transcription occurs in the latter.

Autoradiographic study of RNA synthesis during meiotic prophase in the human oocyte.

The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.

Patterns of RNA synthesis in early meiotic prophase oocytes from fetal mouse ovaries.

In a study of the early meiotic prophase stages of mouse oogenesis from d12 of gestation to 10d post-partum the patterns of RNA synthesis during these stages of oogenesis using H3-uridine incorporation as visualized by light microscope autoradiography are reported. We find that chromosomal RNA synthesis occurs in all stages except early to mid-pachytene, the time of maximum chromosome condensation. Diplotene and dictyate nuclei are the most heavily labelled stages. Nucleolar labelling ceases before leptotene and reappears in late pachytene or early diplotene, even though nucleoli can be identified in all stages except early to mid-pachytene.

High resolution autoradiographic studies of Ehrlich tumour cell nucleoli: Nucleolar labelling after [ 3H]actinomycin D binding to DNA or after [ 3H]TdR or [ 3H]uridine incorporation in nucleic acids

Ehrlich tumour cell nucleoli contain fibrillar and granular components and low electron density areas called “fibrillar centres”. Analysis by high-resolution autoradiography using [ 3H]actinomycin D or [ 3H]TdR reveals that a small amount of DNA is present inside the fibrillar centres. Newly synthesized RNA is also present within the fibrillar centres or at their periphery, already 3 min after the precursor is given. According to these results, RNA is synthesized on DNA in the fibrillar centres. It is possible that the latter contain dispersed genetically active chromatin. These observations add further support to the hypothesis that fibrillar centres have a chromosomal origin and are related to the nucleolar organizers.

Nucleolar activity and RNA metabolism in previtellogenic and vitellogenic oocytes of Xenopus laevis: A biochemical and autoradiographical, light and EM study

Biochemical findings on isolated germinal vesicles and autoradiograms for the light and electron microscopes, after labelling with [ 3H]uridine, have given corroborating results for both previtellogenic and vitellogenic oocytes. Ribosomal RNA (rRNA) is synthesized throughout the previtellogenic period (in oocytes ranging from 10 to 300 μm in diameter), but the density of nucleolar labelling decreases progressively during this period. In the final previtellogenic stage (200 to 300 μm oocytes), it is extremely low and the nucleoli display fibrillar structure; the cytoplasm is almost devoid of ribosomes. Autoradiographical and biochemical methods have permitted quantitative estimations of labelled RNA in previtellogenic oocytes about 100 μm in diameter. Ribosomal (nucleolar) RNA represents only 5% of the labelled RNA which accumulates in the germinal vesicles after several hours of [ 3H]uridine incorporation. The situation is reversed when vitellogenesis begins (oocytes 400 μm in diameter): 30S rRNA makes up more than 40% of the total nuclear label. The many large nucleoli are densely labelled; the cytoplasm is crowded with ribosomes. The morphology of the nucleoli during the previtellogenic stages is consistent with the possibility of inhibition of rDNA transcription during previtellogenesis. This hypothesis is discussed.

An autoradiographic study of tritiated uridine incorporation into the larval ovary of Xenopus laevis.

The in vitro incorporation of 3H-5-uridine into the germinal and somatic cells of the larval ovary of Xenopus laevis has been studied using both light and electron microscope autoradiography. Incubation for only one hour in the presence of precursor revealed that the follicle cells are highly active in rRNA synthesis, whereas substantial oogonial nucleolar labeling was not detected for several hours. Semi-quantitative analysis of high-resolution autoradiograms indicated that the density of silver grains associated with "nuage" in oogonia was almost 4-fold greater than the surrounding cytoplasm. This strongly suggests that a significant amount of RNA is associated with "nuage" at this stage of Xenopus oogenesis, in addition to its well documented protein composition. "Pulse-chase" experiments further suggest that the nuage-associated RNA is stable for at least 24 hours. These results are discussed (within the limitations imposed by the methodology) both in relation to other studies on the composition of nuage in a wide variety of germinal cell types and in the light of growing speculation that nuage and germinal granules are synonymous.

Autoradiographic localisation of 3H-uridine in spinal ganglion neurones of the rat and the effects of methyl mercury poisoning.

The uptake of 3H-uridine into rat spinal ganglion neurones has been followed by autoradiography for up to 48 h after its intravenous injection. Labelling of nucleoli and of nuclei reached a peak within 1 h and then declined. Nuclear labelling returned to background levels by 24 h, but nucleolar labelling was still significant after 48 h. Animals dosed with methyl mercury chloride (7.5 mg/kg daily) showed no change in labelling rate in nucleolus or nucleus after 1, 2, or 4 doses. After 8 doses there was severe reduction in labelling in both nucleolus and nucleus; this amount causes extensive loss of axons, loss of some cell bodies and a marked reduction in amino acid incorporation into proteins. On recovery after a further 8 days, labelling levels returned to normal. It is concluded that at the time when loss of ribosomes occurs from the cytoplasm methyl mercury is more likely to be directly disturbing ribosomal structure than RNA synthesis, for methyl mercury causes marked changes in ribosomal organisation after 4 doses, but disturbances to RNA synthesis do not occur until the 8th dose.

Short-term effects of ACTH on protein synthesis in adrenal cortex cells of young rats

Two units of ACTH were administered intraperitoneally to young 20 gm-rats which received an intravenous injection of L-leucine-3H thirteen min later. ACTH-injected rats, and control rats which received the isotope alone, were killed at 2-, 10-, 30- and 60-min intervals. Electron microscope autoradiographs in control animals showed strong amino-acid uptake at pulse time (2-min) in the cytoplasm of adrenal zona fasciculata cells. Label was shared between the endoplasmic reticulum (ER) and mitochondria, and a lower but still considerable uptake was seen in nucleoli. At first chase time interval (10-min) cytoplasmic labelling declined, while nuclear and nucleolar labelling increased, both changing little thereafter, and there was a 10-30 min Golgi peak. ACTH administration provoked an overall increase in amino-acid incorporation into cytoplasm, nucleus and nucleolus at pulse time, with no changes in the distribution of the reactions among organelles. Intensification of labelling was most evident over nucleoli, the grain density of which was four-times as high as in controls. The short-term increase in ER and mitochondrial protein synthesis observed after ACTH injections was considered to be consistent with the hypothesis that most newly-formed proteins in these cells may be involved in the regulation of steroidogenesis. The marked increase in nucleolar labelling suggested the presence of proteins involved in RNA synthesis.

The RNA polymerase activity of the preimplantation mouse embryo

The activation of the mouse embryo genome has been studied during early cleavage, in vivo. Individual embryos, prepared as whole mounts, were assayed for endogenous RNA polymerase activity. RNA synthesis was detected by autoradiography as the incorporation of [3H]UMP into an acid-insoluble product. No RNA polymerase activity could be detected in the pronuclei of one-cell embryos. Radioactive incorporation was first evident in the nuclei of two-cell embryos. This appeared to be confined to the nucleoplasm and could be abolished by alpha-amanitin but not by low concentrations of actinomycin D. Polymerase activity which was not affected by alpha-amanitin was first detected in the four-cell embryo, predominantly at the peripheries of the nucleoli. Nucleolar labelling increased markedly between subsequent cleavages, reaching a peak in early morulae. In one- and two-cell embryos, label incorporation could be found in the nucleus of the persisting polar body.

Nucleolar RNA synthesis of meiotic prophase spermatocytes in the human testis.

Human meiotic prophase spermatocyte nuclei were studied by electron microscope autoradiography after a 3 hours 3H-uridine labeling pulse, followed by postincubation in non-radioactive medium. In autosomes, 3H-uridine nucleolar labeling reaches a peak during early-middle zygotene prior to the peak labeling of chromosmal RNA species at middle pachytene. Transcription activities of sex chromosomes are inconspicuous when compared with that of autosomes. An increasing condensation of nucleolar-associated chromatin in acrocentric bivalents contributes to the formation of basal knobs in human pachytene spermatocytes. Upon completion of knob formation, nucleolar components segregate and the uptake of 3H-uridine decreases. These findings suggest that the template capability of ribosomal DNA cistrons, located next to the basal knob region, is largely associated with a dispersed state of chromatin whereas increased chromatin condensation is correlated with a restriction of ribosomal RNA transcription.

The mechanism of regulation of fibroblastic cell replication. II. Participation of the nucleoli.

AbstractCultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar 4 cells and in the levels of uptake of 3H‐uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso‐nucleolinar type. The intensity of nucleolar labeling by 3H‐uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre‐ and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar 3H‐uridine incorporation.The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis.During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.

Incorporation of 3 H-uridine into bovine cells doubly infected with the virus of bovine viral diarrhea and a bovine enterovirus.

Tritiated uridine was selectively incorporated into the cytoplasm but not the nucleus of BEK cells infected with bovine enterovirus. This was accompanied by complete shutdown of cellular RNA synthesis. Bovine embryonic spleen cells infected with the BVD virus incorporated3H-uridine into the nucleus and cytoplasm, while uninfected cells showed only nuclear and nucleolar incorporation. Bovine embryonic spleen cells that were preinfected with BVD virus for 20 hours and then superinfected with bovine enterovirus showed complete absence of nuclear and nucleolar labelling in the early stages of superinfection with the bovine enterovirus, but nuclear labelling returned in reduced amounts 8 to 10 hours after superinfection and persisted throughout the remainder of the double infection ending in cell death.

Cytological studies on the inhibition by 5-fluorouracil of ribosome synthesis and growth in jerusalem artichoke tuber slices

Rapid auxin-induced cell expansion in artichoke tuber slices is obtained by aerating the slices in water ("aging") prior to auxin treatment. 5-fluorouracil (5-FU), an inhibitor of ribosomal RNA synthesis in plant cells, markedly inhibits auxin-induced growth only if present in the pre-growth aging period. Autoradiographic studies show that 5-FU given in the aging and/or growth periods reduces the incorporation of RNA precursors into the cytoplasm. Pulse-chase experiments suggest that the reduced cytoplasmic incorporation is in large part due to decreased stability of ribosomal rNA, as nucleolar and chromatin label are only slightly depressed at the end of the pulse. Though the nucleoli continue to incorporate RNA precursors following 5-FU treatment, they lack a distinct granular zone, and appear as homogeneous fibrillar structures under the electron microscope. 5-FU has a parallel inhibitory effect on growth and protein synthesis as shown by (3)H-leucine studies during the growth period. Electron-microscope studies show that treatment with 5-FU causes decreased numbers of ribosomes and rough endoplasmic reticulum. The results suggest that the ribosomes and rough endoplasmic reticulum formed during aging are important in obtaining subsequent rapid auxin-induced expansion. The new ribosomes serve in part to replace pre-existing ribosomes present at the time of excision, which from electron microscopic evidence from 5-FU treated tissue, appear to slowly disappear.

Autoradiographic studies on the kinetics of nuclear-cytoplasmic transfer of RNA in blast cells of acute leukaemia.

Autoradiographic investigations on normal PHA- timulated lymphocytes and on blast cells of acute leukaemia were carried out in order to evaluate the nuclear-cytoplasmic shift of ³Huridine and the degree of nucleolar labelling with the same radioactive precursor. The results obtained indicate that, in contrast with PHA blast cells, a variable proportion of leukaemic blasts, even after prolonged periods of incubation, show a delayed transfer of uridine to the cytoplasm, as well as a reduced concentration of label in the nucleoli. The implications of these findings are discussed, also in the light of recent biochemical acquisitions