Nucleolar necklace formation in response to hemolymph ecdysteroid peaks.

Previous work on the last (fifth) larval stadium of Calpodes showed two phases of elaboration of epidermal nucleoli correlated with RNA synthesis, the first after ecdysis at the beginning of the intermolt and the second near the end of the stadium prior to molting. Both phases followed periods of elevated hemolymph ecdysteroid. The demonstration of four hemolymph ecdysteroid peaks and an improvement in the bismuth-staining procedure for nucleoli has prompted further study of nucleolar changes in relation to hemolymph edcysteroids. We have found that three of the four ecdysteroid peaks (I, II and IV) are followed by nucleolar changes. The exception is the commitment peak (III) for which there is no corresponding nucleolar change. The three nucleolar cycles are similar in their essential features. An intercycle nucleolus consists of one or a few irregularly shaped particles that become more densely stained and condense into a knot at the beginning of each cycle. The knot unfolds into a necklace which beomes beaded as it elongates to a length of about 23 mum. Cells have one or two, rarely more, necklaces presumably depending on their ploidy. At the end of the cycle the necklaces contract, becoming coarser and fragmented before they condense to the intercycle condition of central irregular cores. Whereas nucleolar necklaces are a general response to hemolymph ecdysteroids, mitoses are locally determined and are imposed over other nuclear activities at any time in the third nucleolar cycle.     

The pairing of nucleolar patterns in an epithelium as evidence for a conserved nuclear skeleton

The nucleoli in epidermal cells of fifth instar Calpodes and Manduca larvae undergo three cycles of unravelling into necklaces with condensation back to a dense particle or particles. Mitosis occurs before the first cycle and at the beginning of the third cycle. In spite of the formation and condensation of these nucleolar necklaces the nucleoli in the intercycle condition all have paired patterns. Adjacent pairs of nuclei have nucleoli that resemble one another in the number of their component particles, the shape and size of the particles and sometimes in their arrangement as mirror images. The paired patterns begin at mitosis and reform after necklace elongation and condensation. The patterns are presumed to reflect a nuclear skeleton that is paired from mitosis and conserved until the next mitosis. The nuclear pairing is related to the retention of mid bodies by sibling cells so that the epidermis is a collection of minimal two-cell syncytia.     

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation cells.     

SPHEROIDAL AND RING NUCLEOLI IN AMPHIBIAN OOCYTES : Patterns of Uridine Incorporation and Fine Structural Features

In maturing oocytes of the newt Triturus viridescens, the nucleoli undergo a series of morphological changes that are very similar to those described by Callan for the axolotl, Ambystoma mexicanum. The nucleoli first assume the form of spheroids which then become extended into ring or necklace shapes that are DNase-sensitive; in mature oocytes the nucleoli revert to a spheroidal form. Short term in vitro incorporation studies with uridine-³H on both species show that RNA synthesis occurs in a restricted, eccentric portion of the spheroidal nucleoli, thereby producing an asymmetrical pattern of labeling. In the ring forms, however, the localization of the radioactivity suggests that synthesis takes place symmetrically throughout their entire length. The changes in nucleolar morphology apparently reflect the fact that the component DNA has undergone a redistribution from a localized region in the spheroidal nucleoli to an extended circle in the rings; the patterns of uridine-³H incorporation, therefore, parallel the distribution of DNA in both the spheroidal and the ring nucleoli. Ultrastructurally, the nucleoli contain a fibrillar component that corresponds in position to that of the DNA. The typical spheroidal nucleolus consists of a fibrillar core situated eccentrically and surrounded by a hull of granular, ribonucleoprotein material. The ring nucleoli are composed of a central fibrous region that is ensheathed all around its circumference by a layer of similar granular material. This granular substance is thicker at intervals along the length of the rings, representing the "beads" of the necklaces.     

The nucleolus during epidermal development in an insect

The fifth stadium of Calpodes has two phases of epidermal cell development corresponding to preparation for intermoult and for moult syntheses. Both phases begin with a period of elevated RNA synthesis and the elaboration of a multilobed nucleolus. The apparent number of nucleoli changes from about two to eight and back to two again within the few hours of elevated RNA syntheses. The nucleolar changes are preceded by elevated litres of haemolymph ecdysteroid. During the two periods of activity, alveoli in the matrix of the nucleoli contain particles believed to be ribosomal precursors. The staining properties of these granules differ according to size in a way that suggests a developmental sequence. Mature granules are about 20 nm in diameter and do not stain with bismuth. They are found at the periphery of the nucleolus, in the nucleoplasm, at the approaches to and within the nucleopores. Perichromatin granules, believed to be m-RNA precursor packages, are up to 60 nm in diameter, do stain with bismuth and are found at the periphery of chromatin, in nucleoplasm and distorted at the approaches to the nuclear pores to fit within the central channel. During these periods of heightened activity the nuclear envelope contains microvesicles that may be free or attached to either nuclear or cytoplasmic surfaces. The structure is appropriate for the microvesicular transnuclear envelope movement of molecules such as the ecdysteroid believed to initiate the nuclear changes.     

NUCLEOLAR AGING IN TETRAHYMENA DURING THE CULTURAL GROWTH CYCLE

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.     

Nucleolar apparatus in the macronucleus of Didinium nasutum (Ciliata): EM and 3D reconstruction

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.     

Nucleolar reorganization in epithelial cells of the jejunum of the rat

Light microscope studies were made on nucleoli of jejunal epithelial cells of normal rats fixed in OsO₄, glutaraldehyde, F.A.A. and HgCl₂ and stained with basic fuchsin–alkaline methylene blue. Nucleolar reorganization is extensive and clearly resembles the phenomenon of nucleolar segregation. Polymorphous nucleoli of undifferentiated crypt cells show intermingled constituents and stain purple whereas similar nucleoli of definitive absorptive cells show two homogeneous components–A, stained red and B, stained blue. Cytochemical studies indicate that component A is largely protein and acidophilic and component B is largely nucleic acids and basophilic. These nucleoli become compacted, each forming an amphinucleolus with the two components at opposite poles. Further changes occur along the villi and the components generally separate to form a condensed plasmosome and a diffuse karysome. Extruded cells show nucleolar fragmentation. Electron micrographs of OsO₄ material were used in preparation of wax models. These, along with electron micrographs of glutaraldehyde material stained with uranyl acetate and lead citrate, clearly illustrate and duplicate light microscope findings and strongly resemble nucleolar segregation produced by antimetabolites. Cells of the villi with reorganized nucleoli do not undergo mitosis whereas undifferentiated crypt cells do so. Furthermore, nucleolar reorganization is correlated with aging since it begins in crypt cells and culminates in senescent cells at the tips of the villi. A review is given of the extensive evidence showing that, in the intestine certain functional changes occur similar to those demonstrated in experimental nucleolar segregation. These include gradually changing patterns of DNA, RNA and protein synthesis as well as enzyme activity. The accompany and probably result from nucleolar reorganization.     

Nucleolar DNA organization in the interphase nuclei of mouse fibroblasts.

The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.     

Nucleolus and large nucleolar aggregates of condensed chromatin in interphase nuclei of L 929 cells

Summary Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase.In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.     

扁豆绒毡层发育的超微结构研究

应用透射电镜对扁豆绒毡层发育过程进行了研究,主要结果如下：１）首次发现扁豆绒毡层在发育过程中,经历了二交胞质重组（第一次始于减数分裂末期Ⅱ,第二次始于小孢子发育早期）,使绒毡层细胞的活动呈现３个高峰期（即小孢子母细胞减数分裂期、小孢子四分体期一小孢子早期、小孢子晚期－二胞花粉中期）。２绒毡层细胞的分泌作用有３种形式（渗透分泌、胞吐分泌和自溶）。３．首次观察到绒毡层细胞的内切向壁和径向壁经历了两个周     

Effect of gamma irradiation on nucleolar activity in root tip cells of tetraploid Triticum turgidum ssp. durum L

Ionizing irradiation induces positive or negative changes in plant growth (M1) depending on the amount of irradiation applied to seeds or plant parts. The effect of 50–350 Gy gamma irradiation of kernels on nucleolar activity, as an indicator of metabolic activity, in root tip cells of tetraploid wheat Triticum turgidum ssp. durum L. cv. Orania (AABB) was investigated. The number of nucleoli present in nuclei and micronuclei as well as the mitotic index in the different irradiation dosages was used as an indicator of the cells entering mitosis, the chromosomes with nucleolar organizer regions that are active as well as chromosome doubling in the event of unsuccessful mitotic division. Nucleolar activity was investigated from 17.5 to 47.5 h after the onset of imbibition to study the first mitotic division and its consequences on the cells that were in G₂ and G₁ phases at the time of gamma irradiation. Untreated material produced a maximum of four nucleoli formed by the nucleolar organizing regions (NORs) on chromosomes 1B and 6B. In irradiated material, additional nucleoli were noted that are due to the activation of the NORs on chromosome 1A in micronuclei. The onset of mitosis was highly significantly retarded in comparison to the control due to checkpoints in the G₂ phase for the repairing of damaged DNA. This study is the first to report on the appearance of nucleoli in micronuclei as well as activation of NORs in the micronuclei that are inactive in the nucleus and the effect of chromosome doubling on nucleolar activity in the event of unsuccessful mitotic division.

     

Three-dimensional organization of nucleoli of Didinium nasutum (Ciliata)

A comparative study of nucleolar organization in the somatic nuclei of the ciliate Didinium nasutum was carried out using 3D reconstruction on the basis of serial ultrathin sections. Recently fed interphase ciliates, starved interphase ciliates and cysts were studied. The nucleoli at the interphase stage were shown to have a complex architecture: the fibrillar component forms a complicated network, the granular component is located inside of it. It was shown that nucleoli, which look like individual structures in single sections, are in fact parts of branched nucleolar networks. A 30-h starvation doesn't lead to disintegration of these networks. However in the starved cells the granular component becomes more dense and vacuolized. In the fed ciliates there are many holes in the fibrillar component, whereas in starved ones the fibrillar component is virtually devoid of them. These holes can be proposed to ensure the transport of newly synthesized rRNP. The nucleolar networks didn't occur in D. nasutum cysts. Nucleoli in the cysts look like small individual structures, mainly consisting of fibrogranular component.     

Three-dimensional structure of the ciliate Didinium nasutum nucleoli

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.