Peculiar behavior of the nucleolus and appearance of cytoplasmic nucleolus-like bodies in the roottip meristems ofBrodiaea uniflora Engl. Grown at low temperature

Summary Cytoplasmic nucleolus-like bodies, which showed vast variation both in size and in number per cell, were sometimes found in the telophase cells ofBrodiaea uniflora. When the plants were grown at low temperature, the frequency of telophase cells bearing cytoplasmic nucleolus-like bodies increased with the lapse of days. In contrast, growth at moderate temperature reduced this frequency. Prolonged exposure of the plants to low temperature caused peculiar phenomena concerning the behavior of the nucleolus and nucleolar material: 1. retention of nucleolar remnants at high frequency in metaphase, 2. pulverization of the nucleolar remnants into a great number of minute, fluffed fragments during metaphase, 3. appearance of dot-like nucleolar material in anaphase, and 4. appearance of nucleolus-like bodies, sometimes more than 10 m in diameter, in telophase. All these structures were strongly impregnated with silver. Electron microscopy revealed that both the nucleolar remnant and the nucleolus-like body consisted primarily of fibrils. Our observations clearly demonstrate that the nucleolus-like bodies are derived from the fibrillar component of the nucleolus and are formed by fusion of dot-like nucleolar material during anaphase.     

The karyosphere during late oogenesis in Rana ridibunda.

The organization of the nucleus in the oocytes of Rana ridibunda was examined during late diplotene at the light and electron microscopic level. At this stage the chromosomes are relatively condensed and assembled in the centre of the nucleus, constituting a karyosphere. The chromosomes here are associated with the central "protein sphere" (15--20 microns in diameter), obviously at their telomeres. Numerous nucleoli are accumulated around the chromosomes, forming a karyosphere capsule and contain segregated fibrillar and granular components; structures resembling perinucleolar chromatin and fibrillar bodies (spherules) are associated with the nucleoli. Granules 30 to 40 nm in diameter are seen to surround the fibrillar spherules. "Nucleolus-like bodies" consisting of granules 10 to 15 nm in diameter which are embedded in finely fibrillar material are often associated in contact with the chromosomes. The central sphere is an accumulation of annular structures similar to those of the pore complexes of the nuclear envelope. These structures are bound to the chromosome material, the "nucleolus-like bodies" and the fibrillar bodies. A participation of "nucleolus-like bodies" in the formation of the central sphere is suggested. A possible role of the nuclear protein matrix in the construction of the karyosphere elements is discussed.     

Ultrastructure of the nuclear apparatus of the infusorian Loxodes magnus. I. The macronuclei, macronuclear anlagen and the interphasic micronuclei]

The nuclear apparatus of Loxodes magnus Stokes (Holotricha) consists of numerous macronuclei which belong to the diploid type and never divide, and of numerous micronuclei. No nuclear groups exist; individual nuclei often lie in cytoplasmic islets surrounded by large lacunae of the smooth endoplasmic reticulum. Interphasic micronuclei have two-membraned envelopes with numerous pores, usually lined at the cytoplasmic side with a layer of vacuoles, channels, or flattened vesicles of the smooth endoplasmic reticulum. The chromatin of the micronuclei consists of anastomosing threads, 0.1--0.2 mum wide, between which several nucleolus-like bodies of microfibrillar structure occur. Adult macronuclei have a similar nuclear envelope and a similar system of vacuoles, channels, and flattened agranular cisternae outside it. The macronucleus contains a single large composite nucleolus with 3 or 4 fibrillar cores inside the common granular cortex. The fibrillar cores are pierced by channels containing nucleolar organizers in the form of strands of condensed chromatin. The peripheral zone of the macronucleus is filled with decondensed chromatin fibrils and contains a number of small chromocenters and several aggregates of RNP granules. No protein inclusions (spheres) have been observed in Loxodes macronuclei. The macronuclear anlagen, developing in the cycle of every cell division, show progressive decondensation of the chromosomes and formation of several nucleoli, each with its own organizer. Later on, the nucleoli fuse into a single nucleolus. The small chromocentres are the last to form.     

The ultrastructure of the rabbit oocyte at the bilaminar follicle stage]

The electron microscope study of the nucleus and organoids of the rabbit oocytes cytoplasm during growth showed nucleoluslike bodies (RNP-granules) on the lampbrushen chromosomes to reach their maximal size at the stage of bilaminar follicle. The RNP-granules differ from the nucleoli by the time of their occurrence cytochemical characteristics, and by their ultrastructural pattern. Throughout the bilaminar follicle stage four components may be seen in the oocyte nucleolus: a dense fibrillar framework around the vacuoles, islets of the granular mass loosely dispersed, and electron dense fibrillar elements filling up the numberous electrontransparant vacuoles. The nucleolus-like bodies are round in shape and have no vacuoles, consisting to two components only: distinctly outlined granules, and weakly developed fibrillar component. The nuclear envelope is seen blebbing. Separation of two nuclear membranes forms a pocket-like enlargements of the perinuclear space. The pockets are limited by small regions between the adjacent nuclear pores. The outer membrane may bulge producing lacuma and large channels in the cytoplasm, which are interconnected making a closed branched network extending inside of the cytoplasm. The nuclear envelope is suggested to be involved in formation of the endoplasmic reticulum through the blebbing process.     

Fragmentation of polyploid nuclei in the giant cells of the rat trophoblast. I. The ultrastructure of the nuclear fragments

Electron microscope study of the nuclear fragments in the rat trophoblast has demonstrated that the division of the trophoblast giant nucleus results first in the formation of a multinuclear cell. Each nuclear fragment is covered with its own nuclear envelope made of two membranes with numerous pore complexes. The chromatin in these nuclear fragments is condenced with various degrees of condensation, which depends on the step of placenta development, cell differentiation and the degree of nuclear fragmentation. The nuclear ultrastructure in nuclear fragments also depends on the degree of nuclear fragmentation and on the level of chromatin condensation. The nucleolus has no granular component. On large fragments, with lower chromatin condensation the nucleolus is not homogenous being made of fragments of more and of less electron dense fibrilles. Small light lacunae are seen in the nucleolus where chromatin threads and strands pass on. With a high chromatin condensation in the nucleus, round small nucleoli look homogenous being made of moderately electron dense fibrilles. Products of chromosome activity have been found in the nuclear fragments: accumulations of minute granules (d = 15--20 nm), perichromatinous granules (d = 35--40 nm), and fibrillar nucleolus-like bodies. In the multinuclear cell, made as the result of fragmentation of the initially giant nucleus, all the small nuclei are first arranged very close to each other, so that the contours of the neighbouring nuclei coincide.     

Nuclear ultrastructure of the oocytes from human antral follicles. Formation of the karyosphere

The organization of the nucleus in the oocytes from human antral follicles was examined at the electron microscopic level. At this time all the chromosomes are aggregated around an inactivated nucleolus forming a karyosphere 5-7 micron in diameter. The nucleolus bears no granular component and consists of densely packed delicate fibrillar material. The peripheral zone resembling a ring 0.5 micron thick is separated in the nucleolus. Nucleolus-like bodies (NLB), consisting of granules 20 nm in diameter embedded in finely fibrillar material, are constantly observed in contact with the chromatin. The eventually formed karyosphere is a complex of intimately interconnecting structures--the nucleolus, chromosomes and NLB. However, the chromatin surrounding the nucleolus does not form a continuous (compact) mass as it is observed at the light microscopic level. It is determined that the human karyosphere is formed during the preovulatory period when the connection between oocyte and follicular cells of cumulus oophorus is lost. The duration of karyosphere existence in the human oocytes, and relation of the karyosphere to the processes of antral follicle atresia are discussed.     

Nucleologenesis: Composition and fate of prenucleolar bodies

A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently reorganize and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.     

A CORRELATED LIGHT AND ELECTRON MICROSCOPE STUDY OF THE NUCLEOLAR MATERIAL DURING MITOSIS IN VICIA FABA

Root meristematic cells of Vicia faba were examined, with both light and electron microscopes, in order to study the behaviour of the nucleolar material during the mitotic process. Under light microscopy, the preprophase nucleolus is seen to consist of a densely stained material in which are embedded several unstained vacuole-like structures of varying size. The electron microscope reveals that the dense nucleolar material is formed of two structurally distinct components, each segregated into irregularly shaped zones blending with one another. One of these components is represented by 150 A granules which, in places, are arranged into thread-like structures approximately 0.1 µ in diameter; the other component apparently consists of fibrils 60 to 100 A in diameter. The large and medium sized intranucleolar vacuoles contain loosely scattered granules and fibrils similar to those just described. The granular and fibrillar components of the denser portion of the nucleolus persist as such during prophase and disperse throughout the nuclear cavity at the time of nucleolar disintegration. After nuclear membrane breakdown, these granules and fibrils, as well as those of the nucleoplasm, mix freely with similar elements already present within the forming spindle. No evidence has been obtained that, during or after nucleolar disintegration, the structural components of the nucleolus become associated as such with the chromosomes to form an external or internal matrix. Our observations suggest the existence, of a matrix substance within late prophase, metaphase, and anaphase chromosomes, the fine structure of which bears strong resemblance to that of their constituent coiled chromonemata. Data are presented, moreover, that indicate that part of this matrix substance, presumably formed at some time during prophase, is released from the chromosomes during their anaphasic movement. A number of observations indicate that the main bulk of the next nucleolus is derived from a prenucleolar fibrillogranular material, arranged into thread-like structures some 0.1 µ in diameter, which collect in the interchromosomal spaces during early and midtelophase. Finally, our data would seem to favour the view that most of this prenucleolar material results from a resumption of the synthetic activity of the early and midtelophase chromosomes rather than from a mere shedding of a preexisting matrix substance.     

Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy

The ultrastructural distribution of proteins B23 and nucleolin in the nucleolus of mouse embryos from the zygote to the early blastocyst has been analyzed by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. In parallel, silver staining of nucleoli was carried out on ultrathin sections. Our results show that the compact prenucleolar bodies at 1- and 2-cell stage as well as the compact residual fibrillar masses observed up to the morula stage, are labelled with the two antibodies. These masses, however, are not stained with silver up to the 4-cell stage. In well-developed nucleoli, the two antibodies co-localize in the dense fibrillar component (DFC) and the granular component (GC) while fibrillar centers (FCs) are devoid of label. On the contrary, silver staining occurs in the FCs and DFC but not in the GC. Our observations suggest that there is no direct relationship between the occurrence of silver staining and the distribution of protein B23 or nucleolin. Moreover, neither the localization of the two above proteins nor silver staining are unequivocally related to the nucleolar activity.     

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

Functional organization of the nucleus in the oogenesis of Chrysopa perla L. (Insecta,Neuroptera)

On the basis of light, autoradiographic (uridine-³H incorporation) and electron microscopic investigation changes of nuclear structures were examined during the oogenesis in Chrysopa perla L. — In early meiotic prophase the oocyte nuclei were found to contain a large body of extrachromosomal DNA. In certain cases the latter splits up into several DNA clumps giving rise to a few (4–7) primary nucleoli, 3–5 in diameter. The primary nucleoli consist of densely packed fibrils 50–100 Å thick. They contain no granular component and are inactive in RNA synthesis. — At the beginning of large growth the extrachromosomal DNA bodies disappear and numerous electron-dense clumps, 0,5–1 in diameter, appear in the nucleus. Instead of the primary nucleoli, the nucleus now contains a great number of ring nucleoli about 0,5–1 in diameter with a granular component (granules are 150 Å). The space between them is filled up with nucleolar strands running from the surface of the ring nucleoli. — At the stage ring nucleoli of uridine^–3 H incorporation into the oocyte nucleus begins. — During later previtellogenesis and at the beginning of vitellogenesis the ring nucleoli disappear and the nucleus is filled with the network of nucleolar strands. Among them there are specific complexes. These consist of electron dense masses, of granular clusters (granules 500 Å in diameter) and large fibrillar electron light bodies. At this stage the nucleus takes the most active part in RNA synthesis. — The process of karyosphere capsule formation was studied by electron microscopy. The capsule was found to be of fibrillar nature; its structure is very peculiar and unlike any known membrane components of the cell. On the basis of cytochemical evidences the characteristics of the capsule are given. — The development of a powerful nucleolar apparatus based on the extrachromosomal DNA and a possible role of the synaptonemal complex and extrachromosomal DNA in formation of the karyosphere capsule is discussed.     

Ultrastructural Changes of the Nucleolus During Cell Cycle in Triticum aestivum

The ultrastructural changes of the nticleolus during cell cycle in common wheat (Triticum aestivum L. ) were studied by an "en bloc" silver-staining method. It was observed that in interphase, the nucleolus was heavily stained, within which fibrillar centres, dense fibrillar component, granular component and nucleolar vacuoles could be identified. A large quantity of argentine fine granules were distributed in the condensed chromatin. Dur-ing prophase, along with the disintegration of the nucleolus and condensation of the chromatin, the larger heavily-stained granules gradually appeared at the periphery of the chromatin. At late prophase, the materials derived from the nucleolus were spread and deposited on the surface of the chromosomes. The silver-stained, larger granules, deriving from the disintegrated nucleolus, accumulated at the periphery of the metaphase chromosomes and formed an uneven and discontinuous "sheath"-like structure. This "sheath"-like structure was also observed at anaphase. In telophase, the silver-stained nucleolar materials were progressively separated from the "sheath' and fused with each other to form prenucleolar bodies, and at last, participating in the formation of new nucleoli. The results showed that the nucleolar materials were transferred directly to the surface of the chromosomes and formed a discontinuous coat, but not incorporated into the interior of the chromosomes. The silverstained granules inside the chromosomes were neither related to the nucleolus nor to the materials from the disintegrated nucleolus.     

Origin of nucleolus-like bodies found in the nucleoplasm and cytoplasm of Vicia faba meristematic cells

Cytohistochemical staining and RNase-gold labelling have been applied to root-tip meristematic cells of Vicia faba to study the origin and biological significance of 2 types of inclusions: one seen in the nucleoplasm and the other in the cytoplasm of early telophase cells. They have been termed "dense bodies" and "cytoplasmic nucleolus-like bodies" (NLB), respectively. Both types of inclusions respond positively to silver staining and ribonucleoprotein (RNP) staining in a similar fashion to nucleolus. Interestingly, the dense bodies label heavily with the RNase-gold complex, as does the nucleolus, while the cytoplasmic NLB have no affinity with the label. In most cases, the dense bodies label more heavily than the nucleolus. Light microscope surveys reveal that the dense bodies sometimes appear to be released from the surface of the nucleolus. On the other hand, prenucleolar material showing the same silver staining and RNP preferential staining characteristics as the dense bodies begin to accumulate on the surface of chromosomes in mid-anaphase. This material does not label with RNase-gold. These data are discussed in terms of the hypothesis that the dense bodies are derived from the nucleolus by direct budding or fragmentation, and the cytoplasmic NLB are composed of prenucleolar material that failed to attach to chromosomes.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.     

The role of interphase prenucleolar bodies in the recovery of the nucleolar structure after reversible hypotonic treatment

Interphase prenucleolar bodies are globular bodies which accumulate in large numbers in the nucleoplasm of cultivated cells after hypotonic treatment and subsequent return to isotonic conditions; detailed studies of the role of these structures in the recovery of the nucleolus have not yet been performed. The limited mobility of interphase pronucleoli within the nucleus has been demonstrated. Exchange of the major nucleolar protein B23 between prenucleolar bodies and the surrounding nucleoplasm, rather than stable binding of this protein to the prenucleolar bodies, has been demonstrated using fluorescence recovery after photobleaching method. Gradual accumulation of B23 in the recovering nucleolus with concomitant disappearance of prenucleolar bodies has been demonstrated.     

Identification and definition of nucleolus-related fibrillar bodies in micronucleated cells

Small nucleolus-related bodies which occur in the nucleoplasm of "micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.     

M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.     

Ultrastructure of Blepharoplast and Other Organelles of the Spermatid in Ginkgo biloba

Both blepbaroplast and osmiophilic globule were characteristic structures to the spermatid of Ginkgo biloba. The blepharoplast of Ginkgo biloba ranged from 3 ～ 4 μm in diameter and consisted of a number of basal centrioles radiating from an electron dense core that contained electron-lucent areas with microtubule structure. Microtubules extended radially from the blepharoplast into the cytoplasm. A large round osmiopbilie globule with a diameter of about 10～20/μm, was located between the blepharoplast and the nucleus, while a filbrillogranular body in the cytoplasm was opposite to the osmiophilic globule. There were numerous mitochondria, plastids, endoplasmic reticulia and dictiosomes in the cytoplasm, particularly around the blepharoplast and the osmiophilic globule of sperm cells. The nucleus of spermatid in Ginkgo biloba was large and roundly elliptical in shape. The large spheroidal nucleolus was the most obvious structure in the nucleus, There were two regions in the nucleolus distinguished by TEM: A ring-shaped granular component, which contained maturing ribosomal precursor particles; and a centrally placed fibrillar component. The nuclear pore complexes in the nuclear envelope were plentiful but not evenly distributed.