Nucleolar Labeling Research Articles

Visualization of the Nucleolus Using 5' Ethynyl Uridine.

Labeling of the nucleolus in Arabidopsis thaliana can be achieved by incorporation of 5'-ethynyl uridine (EU) into bulk RNA. Although EU does not selectively label the nucleolus, the abundance of ribosomal transcripts results in the predominant accumulation of the signal in the nucleolus. Ethynyl uridine has the advantage of being detected via Click-iT chemistry providing a specific signal and low background. While the protocol presented here employs fluorescent dye and allows visualization of the nucleolus by microscopy, this method can also be used for other downstream applications. Though we tested nucleolar labeling only in A. thaliana, in principle it can be applied to other plant species.

Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions.

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.

Uptake and cellular distribution of nucleolar targeting peptides (NrTPs) in different cell types.

Nucleolar targeting peptides (NrTPs) are a family of cell penetrating peptides (CPPs) derived from crotamine, a rattlesnake venom toxin. They were named NrTPs for their remarkable nucleolus-homing properties and have been studied for their potential as drug delivery vehicles. Live cell microscopy experiments were conducted to monitor NrTP uptake and distribution in different cell types, including primary cells (PBMCs and erythrocytes) and different immortalized cell lines (HeLa, BHK21, BV-173, and MOLT-4). Uptake dependence on cell type (primary vs. immortalized, suspension vs. adherent, cancer vs. healthy cells), peptide concentration and cell viability were evaluated. To gain further insight on the internalization mechanism, uptake kinetics was also monitored. Results showed the uptake and distribution pattern as strongly dependent on peptide sequence, peptide concentration and membrane constituents. Under similar conditions, NrTP6 is more internalized than NrTP1, NrTP2 and NrTP5. Additionally, while internalization of NrTP7 and NrTP8 may cause cytotoxicity, NrTP6 is noncytotoxic. Higher peptide concentrations can be correlated to nucleolar targeting, although even at low concentrations a residual number of cells reveal positive nucleolar labeling. NrTPs were successfully internalized into all cell types tested except erythrocytes.

Localization of Nopp140 within mammalian cells during interphase and mitosis

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.

Nucleolar marker for living cells

In the recent molecular and cell biological research, there is an increasing need for labeling of subcellular structures in living cells. Here, we present the use of a fluorescently labeled cell penetrating peptide for fast labeling of nucleoli in living cells of different species and origin. We show that the short peptide with ten amino acids was able to cross cellular membranes and reach the nucleolar target sites, thereby marking this subnuclear structure in living cells. The treatment of cells with actinomycin D and labeling of B23 protein and fibrillarin provided evidence for a localization to the granular component of the nucleolus. The fluorescently conjugated nucleolar marker could be used in combination with different fluorophores like fluorescent proteins or DNA dyes, and nucleolar labeling was also preserved during fixation and staining of the cells. Furthermore, we observed a high stability of the label in long-term studies over 24 h as well as no effect on the cellular viability and proliferation and on rDNA transcription. The transducible nucleolar marker is therefore a valuable molecular tool for cell biology that allows a fast and easy labeling of this structure in living cells.

5 NUCLEOLAR DEVELOPMENT REQUIRES TRANSCRIPTIONAL ACTIVITY DURING PORCINE EMBRYONIC GENOME ACTIVATION

The development of a functional nucleolus accompanying the major embryonic genome activation (EGA) is considered a marker for embryo quality and viability. However, the use of this marker is limited by the lack of accurate knowledge of the biology of embryonic nucleologenesis. The objective of this study was to elucidate the role of RNA polymerase I (RPI) and total transcriptional activity, reflecting EGA, for nucleologenesis in in vivo-developed porcine embryos. Late 4-cell-stage embryos were cultured in the absence (control) or presence of actinomycin D (AD; 0.2 �g mL-1, 3 h for RPI inhibition; 2.0 �g mL-1, 3 h for total transcriptional inhibition). Late 2-cell-stage embryos were cultured to the late 4-cell stage with 0.2 �g mL-1 AD (long-term inhibition) to prevent EGA. Embryos were fixed at the late 4-cell stage and processed for RT-PCR (de novo synthesized rRNA), autoradiography (ARG, following culture with 3H-uridine for the last 20 min before fixation), TEM, FISH (probe-labeling rRNA and rDNA), silver staining of nucleolar proteins, and immunofluorescence for RPI. Control embryos displayed typical extranucleolar and nucleolar ARG labeling, fibrillo-granular nucleoli, and focal RPI localization signaling de novo rRNA synthesis in functional nucleoli, confirmed by RT-PCR. All nuclei showed large FISH clusters (rRNA and rDNA) that in 88% of the cases were co-localized with large foci of silver-stained nucleolar proteins. After RPI inhibition, only extranucleolar ARG labeling was detected and, instead of fibrillo-granular nucleoli, a segregated dense-fibrillar component and a granular component, but no fibrillar centers, were observed. RPI was dispersed in all nuclei, the number of nuclei presenting large FISH clusters decreased to 40%, and only 42% of nuclei showed nucleolar proteins localized to large foci. After total transcriptional inhibition and long-term inhibition, the nuclei did not display any ARG labeling and presented inactive nucleolus precursor bodies indicating lack of rRNA (RT-PCR) and total RNA synthesis. However, 40% of the nuclei in both groups presented large FISH clusters of rRNA. This rRNA is considered as partially processed residues of maternally inherited molecules, and their clustering is most likely independent of EGA. Inhibition of transcriptional activity at the time of EGA caused the dispersion of RPI (de novo synthesized) but did not influence the localization of silver-stained nucleolar proteins to large foci (41%). On the other hand, EGA inhibition caused the lack of RPI labeling and hampered the localization of nucleolar proteins to foci. Differences between these 2 groups could be due to the activation of RNA polymerase II before the 3-h AD treatment. In conclusion, RPI transcription and de novo protein synthesis are required for formation of functional nucleoli. However, the clustering of maternally inherited nucleolar transcripts is independent on transcriptional activity at the time of EGA. Failure in constituent RNA polymerase activation during EGA leads to pattern-specific changes in nucleologenesis, which may serve as a marker for early embryo quality.

DADLE induces a reversible hibernation-like state in HeLa cells

[D-Ala(2)-D-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.

A higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies.

Transient transfection of HeLa cells with a plasmid encoding the full-length human fibrillarin fused to a green fluorescent protein (GFP) resulted in two major patterns of intensity of the nucleolar labeling for the chimeric protein: weak and strong. Both patterns were maintained in fibrillarin-GFP expressing cells after fixation with formaldehyde. When the fixed fibrillarin-GFP expressing cells were used for immunolabeling with antibodies to fibrillarin, only the nucleoli with a weak GFP-signal became strongly labeled, whereas those with the heavy signals were only lightly stained, if at all. A similar pattern was observed if the cells were immunolabeled with antibodies to GFP. These observations suggest that an increase in antigen accumulation within the nucleolus, which could take place under various physiological or experimental conditions, could prevent the antigen from being recognized by specific antibodies. These results have implications regarding contradictory data on localization of various nucleolar antigens obtained by conventional immunocytochemistry.

Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis condensin complex in interphase cells.

Cell cycle dynamics and localization of condensins--multiprotein complexes involved in late stages of mitotic chromosome condensation--were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to beta-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF, fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.

Spatial dynamics of rRNAs

The understanding of the functional organization of a compartmentalized organelle such as the nucleus constitutes one of the major challenges in cell biology today. An essential step towards this goal is the study of the spatial dynamics of RNA synthesis and maturation. The in vivo dynamics of nascent RNAs have been commonly investigated by the labeling of transcripts with 3H-uridine, followed by autoradiographic detection. Because of the drawbacks inherent to this technique, an immunocytochemical technique, using bromodeoxyuridine 5′-triphosphate (BrUTP) as an analog, has been developed 1xVisualization of focal sites of transcription within human nuclei. Jackson, D.A et al. EMBO J. 1993; 12: 1054–1065See all References, 2xFluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. Wansink, D.G et al. J. Cell Biol. 1993; 122: 283–293Crossref | PubMedSee all References, 3xNonisotopic ultrastructural mapping of transcription sites within the nucleolus. Dundr, M and Raska, I. Exp. Cell Res. 1993; 208: 275–281Crossref | PubMedSee all References. However, cell permeabilization or microinjection techniques, which are employed to ensure the intracellular penetration of BrdU, disturb cell integrity. Consequently, although this technique has allowed the visualization of nucleolar labeling in the vicinity of the polymerase, it has not permitted the observation of nascent rRNA transport. To improve this approach, we have used the FuGene-6 transfection reagent for the introduction of BrUTP (4xSee all References4). By improving cell preservation in this manner, both extranucleolar RNA transcripts (sensitive to α-amanitin) and ribosomal RNAs (sensitive to actinomycin D) have been rapidly and simultaneously detected. Furthermore, the transport of nascent RNAs was visualized both within the nucleus and from the nucleus to the cytoplasm. By using pulse–chase experiments, three-dimensional reconstructions from confocal sections, and electron microscopy analysis of ultrathin sections, the topological and spatial dynamics of rRNAs within the nucleolus were revealed. Our data demonstrate that rRNA transport does not involve tracks. By contrast, it follows a volumic, well-defined pathway from multiple points that extend from nucleolar speckles towards the periphery of the nucleolus. This migration can be summarized by four typical labeling patterns (Fig. 1Fig. 1). As assessed by double-labeling experiments with nucleolar proteins involved in rRNA synthesis or maturation, these patterns could correspond to different functional domains. The dynamics of rRNAs within the nucleolus are similar to those of some polyadenylated mRNAs within the extranucleolar space 5xMovement of nuclear poly(A)RNA throughout the interchromatin space in living cells. Politz, J.C et al. Curr. Biol. 1999; 9: 285–291Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all References5. Our BrUTP incorporation technique, combined with three-dimensional visualizations and tomographic analyses, should aid significantly our understanding of the functional, volumic organization of the cell nucleus.Fig. 1Localization of bromodeoxyuridine 5′-triphosphate (BrUTP)-labeled rRNAs during their movement from initial sites of transcription to the periphery of the nucleoli. HeLa cells, treated with α-amanitin to inhibit extranucleolar transcription, were transfected by lipofection for 15 min with BrUTP-FuGene-6 complexes and then cultured for various lengths of time before being fixed. Bromo-rRNAs were detected by secondary FITC-coupled antibodies. For each z series, 15-30 optical sections were recorded with a 0.25 μm z-step by exciting the fluorophore at 488 nm and by the simultaneous collection of phase-contrast images. Files were processed using the Analyze software. A surface-visualization procedure was used for each reconstructed volume 6xElectron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization. Heliot, L et al. Mol. Biol. Cell. 1998; 8: 2199–2216CrossrefSee all References6. Results show either a single optical section (a, c, e, g) or a three-dimensional reconstruction of the whole volume (b, d, f, h). Insets in (b), (d) and (f) show a higher magnification of three selected areas. Bar, 5 μm [1 μm for insets in (b) and (d), 2.5 μm for inset in (f)]. The nuclear limits, determined by phase contrast, are represented by dotted lines. A time-course of BrUTP incorporation appeared as a radial flow starting simultaneously from numerous transcription sites, then growing into concentric rings, which finally fused together as they progressed towards the nucleolar periphery.View Large Image | Download PowerPoint Slide

Transient Nucleolar Localization Of U6 Small Nuclear RNA InXenopus LaevisOocytes

Recent studies on the 2'-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

Changes in nuclear localization of An3, a RNA helicase, during oogenesis and embryogenesis in Xenopus laevis.

The immunolocalization of An3 protein, an ATP-dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti-An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti-Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti-Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti-Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3-positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 micrograms/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 micrograms/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA.

Effect of radiation on the functioning of neuron chromatin of mice exposed in the zone of the Chernobyl nuclear power plant

Circadian fluctuations in the activity of endogenous RNA polymerases were examined in the nuclei ofLv spinal ganglion neurocytes, spinal α-motoneurons, and in neurons of the sensorimotor and visual cortex of mice exposed to radiation in the zone of the Chernobyl power plant. The circadian organization of the functioning of the genetic apparatus of neurons in various compartments of the nervous system was altered in irradiated mice. A shift of the acrophases of nucleoplasmic and nucleolar labeling in the course of the 24-hour cycle was observed after a 40-day exposure under conditions of a high radioactive background. The degree of synchronization of the level of transcription of ribosomal and structural genes of the studied cell populations diminished. The mesors and amplitudes of circadian rhythms of chromatin matrix activity decreased. The status of the histone component of chromatin also changed in exposed animals.

Nuclear fine structure and transcription in early goat embryos

Early preimplantation goat embryos were investigated for the onset of major gene transcription by fine-structure morphology and autoradiographic detection of (5- 3H)uridine incorporation. Complex nuclear bodies were already seen in early pronuclei but only in the 16-cell embryos did these methodologies offer clear-cut evidence of the already fully-developed nucleolar structure as well as of nucleoplasm labeling. Nucleoplasm labeling, which is supposed to detect the onset of major transcription, was absent in the pronuclear and the 2- to 4-cell embryos. The first (5- 3H)uridine incorporation was detected in the 8-cell stage nuclei nucleoplasm, followed by nucleolar labeling. Based on this evidence, we found that in comparison with other ruminants, there is no striking difference in the timing of the shift in control from the maternal to the embryonic genome in the development of the early goat embryo.

Factors involved in nuclear reprogramming during early development in the rabbit

Mechanisms of nuclear reprogramming and assessment of potential malfunctions that could be deleterious for development were evaluated in rabbit zygotes, parthenotes, and nuclear transfer embryos by analysis of DNA replication, nucleolar fibrillarin label, and localization of nuclear material reactive to the MPM-2 antibody. Nuclear transfer embryos were derived from G1/early S-phase donor nuclei and MII oocytes. In nuclear transfer embryos, DNA rereplication was likely to have occurred because label was incorporated, possibly in the centromeric regions of the chromosomes, prior to premature chromosome condensation and again following pronuclear formation. In parthenotes, DNA replication began very late in the cell cycle, which may be due to deficiencies in the artificial activation stimulus. The presence of fibrillarin label in the nucleolus was used as an indication of nucleolar transcriptional activity. Fibrillarin label was absent in embryos of all types up to the 16-32-cell stage. Although fibrillarin reappeared in nuclear transfer and parthenote embryos at the appropriate stage, not all blastomeres showed label indicating impaired development in these embryos. Labelling of phosphorylated epitopes by MPM-2 antibody showed a change in pattern of labelling during early development. Early cleavage stage embryos did not exhibit labelling over the spindle poles as did blastomeres from 32-cell embryos and tissue culture cells. All cell types exhibited labelling during interphase as dots located primarily over the nucleus in blastomeres from 32-cell embryos and in tissue culture cells, together with cytoplasmic label in embryos at early cleavage stages. Nuclear transplant embryos had a normal pattern of MPM-2 label. In contrast, the appearance of MPM-2 label in parthenotes depended on the type of calcium stimulation. These results demonstrate defects in DNA synthesis, nucleolar activity, and specific phosphorylation events, likely resulting from an improper activation stimulus and chromosome condensation in the transplanted nucleus.

An Experimental Approach to the Study of AG-NOR Proteins.

Purified polyclonal antibodies have been obtained against 100KD and 36KD Ag-NOR proteins. These two antibodies have been used to study the immunolocalization of both proteins on different cell types subjected to various conditions of cell activity and to observe a nucleolar labelling with anti-36kDa Ag-NOR protein antibody evenly distributed over the dense fibrillar component and granular component, and with anti-100kDa mainly localized to the dense fibrillar component. After actinomycin D treatment, immunolabelling with both antibodies was found restricted to the granular zone of segregated nucleoli. Moreover, to further study the functional role of these proteins, we have used an electroporation system to introduce both antibodies into the nuclei of living cells, and have clearly detected a different nucleolar behaviour. The combination of these techniques has been shown to be an important tool to deepen the knowledge of the nucleolus and has allowed us to propose that the difference in nucleolar localization of the major 36kDa and 100kDa Ag-NOR proteins is the consequence of a different role of both proteins in the synthesis and processing of rRNA.

Nucleolar organizer region score and Ki-67 labelling index in high-grade gliomas and metastatic brain tumours.

Sixteen cases of malignant brain tumours comprising 6 anaplastic astrocytomas, 3 glioblastoma multiforme, 1 medulloblastoma and 6 metastatic brain tumours were investigated independently by a silver colloid method for nucleolar organizer regions (NORs) and an immunohistochemistry using a monoclonal antibody against a nuclear antigen. Ki-67, in proliferating cells. The correlation between the mean number of NORs and the percentage of Ki-67 labelled cells (Ki-67 labelling index) was examined. In addition, four normal brain tissue samples without neoplastic cells were stained for NOR. The mean number of NORs in these malignant brain tumours was significantly greater than that in normal astrocytes (p less than 0.001). Moreover, both the mean number of NORs and the Ki-67 labelling index in metastatic brain tumours were significantly greater than those in high-grade gliomas (p less than 0.001). The Ki-67 labelling index and the mean number of NORs in malignant brain tumours including metastatic brain tumours were found to be linearly related (r = 0.86). These results suggest that the proliferative potential of malignant brain tumours could be evaluated by NOR score as well as Ki-67 labelling index and that such indices provide clear discrimination between high-grade gliomas and metastatic brain tumours.

Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.

Autoradiographic Study of RNA Synthesis During Secondary Rhizoid Cell Differentiation in Pteris vittata

The pre- and postmitotic stages of rhizoid cell differentiation in Pteris vittata L. were pulsed in radioactive uridine for 30 min and the data analyzed quantitatively by autoradiography. The total grain count rises from 594 in the nondifferentiating cell to 1,369 in the corona stage; increases to 2,745 in the internally segmented rhizoid cell (IRC) and to 2,779 in the wheel; and then drops to 1,306 in the protrusion and to 1,261 in the early rhizoid. The significant increase in nucleolar labeling in the corona reflects an increase in ribosomal RNA synthesis for mitosis-associated syntheses. The two-fold increase in grains over the cellular compartments of the IRC and wheel reflects an increase in RNA synthesis in these postmitotic stages. The similarity of labeling profiles between these stages suggests that they are not far enough separated to show a difference in their roles. The decline in total grain counts in the protrusion and early rhizoid stages to half the maximum levels suggests that an overall decrease in RNA synthesis is attended by a shift from a differentiating phase to a steady-state growth system.

AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING SECONDARY RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA

The pre‐ and postmitotic stages of rhizoid cell differentiation in Pteris vittata L. were pulsed in radioactive uridine for 30 min and the data analyzed quantitatively by autoradiography. The total grain count rises from 594 in the nondifferentiating cell to 1,369 in the corona stage; increases to 2,745 in the internally segmented rhizoid cell (IRC) and to 2,779 in the wheel; and then drops to 1,306 in the protrusion and to 1,261 in the early rhizoid. The significant increase in nucleolar labeling in the corona reflects an increase in ribosomal RNA synthesis for mitosis‐associated syntheses. The two‐fold increase in grains over the cellular compartments of the IRC and wheel reflects an increase in RNA synthesis in these postmitotic stages. The similarity of labeling profiles between these stages suggests that they are not far enough separated to show a difference in their roles. The decline in total grain counts in the protrusion and early rhizoid stages to half the maximum levels suggests that an overall decrease in RNA synthesis is attended by a shift from a differentiating phase to a steady‐state growth system.