Nuclear structures in the late oogenesis of Rana temporaria. III. Electron microscopic studies]

The ultrastructural organization of the vitellogenic oocyte nucleus (stage VI, according to Duryee, 1950) was studied in normal and in vitro hormone-stimulated maturing oocytes of Rana temporaria. At this stage, numerous nucleoli are gathered around the knot of highly contracted chromosomes (the karyosphere) thus making the karyosphere capsule. Light microscope observations reveal three zones in the capsule: a central fibrous zone separating the chromosomes from the nucleoli, a middle zone, consisting of numerous nucleoli and a distinct fibrous componen; in addition a fibrous zone on the capsule periphery is seen. The nucleoli are fibrillar, bear no proribosomal granules and do not synthesize RNA. This period is characterized by an intensive fragmentation and segregation of the nucleolar material. Numerous micronucleoli and nuclear bodies occur in the nucleus. The nucleoli are normally compound and irregular in shape to become spherical in hormone-stimulated maturing oocytes. In the central fibrous zone of the capsule, separating the chromosomes from the nucleoli, some peculiar abundant accumulations of annuli were detected lacking the membranes component. Annuli are linked with the fibrous material and are regularily packed making peculiar pseudomembranes (PMM). The chromatin is connected with PMM directly. In the middle zone of the capsule, accumulations of PMM are also seen, though less abundant and less regularly packed; along with annuli, membranous areas of various size and form are met in PMM. PMM are connected with the micronucleoli with filaments 20 nm thick. In the peripheral zone of the capsule, a variety of membranous structures is detected: intranuclear annuli lamellae, component of the capsule consists of different membranous and pseudomembranous (with annuli) structures. A question of the contribution of the chromatin material in the formation of the fibrous capsular component (PMM and membranous structures) is discussed.     

Studies on nucleoli in rosetting T and B lymphocytes of the human peripheral blood

The incidence of various nucleolar types was studied in human rosetting lymphocytes to provide an information on nucleolar types present in T and B lymphocytes of the peripheral blood. The results clearly demonstrate that both T and B lymphocytes of the peripheral blood mostly contain ring shaped nucleoli ("resting nucleoli") and less frequently other nucleolar types such as nucleoli with nucleolonemata or compact nucleoli ("active nucleoli") and micronucleoli ("inactive nucleoli"). Since all known nucleolar types and particularly micronucleoli may be observed in both T and B lymphocytes, nucleoli in these cells cannot indicate the type or origin of these cells but simply the state of the nucleolar RNA synthesis.     

Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria

We have examined the distribution of RNA processing factors in the germinal vesicle (GV) of the common frog Rana temporaria during early vitellogenesis by immunostaining on light- and electronmicroscopic levels and by in situ nucleic acid hybridization. Small nuclear RNPs (snRNP) and factor SC35 involved in pre-mRNA splicing occur in lampbrush chromosome loops and numerous granules 1-3 microns in size. These granules are identical to B snurposomes of Xenopus laevis and Notophtalmus viridescens described earlier (Wu et al., 1991). Some of B snurposomes are attached to homologous loops of lampbrush chromosomes. Immunofluorescent study of Cajal bodies/coiled bodies (CB) showed that sometimes CB have B snurposomes attached to their surface. In this case splicing factor SC35 is found in B snurposomes and B-like inclusions in CB matrix. In CB without attached B snurposomes splicing factor SC35 localizes throughout the whole organelle. Staining of GV spreads with antibodies against nucleolar protein NO38 revealed this protein in CB, nucleoli and micronucleoli. Using in situ nucleic acid hybridization and immunofluorescent staining we have found that on GV spreads from hibernating frogs B snurposomes contact nucleoli. Nucleoli contain snRNP. These data suggest that nucleoli may be storage sites of snRNPs during natural inactivation of RNA synthesis. During winter season in Rana temporaria GV nucleoli become compacted and a number of micronucleoli (less than 2 microns) dramatically increases. Analysis of micronucleoli showed that they contain rRNA, protein NO38, trace amount of U3 small nucleolar RNA and do not contain fibrillarin, involved as U3 in pre-rRNA processing. We suggest that decrease of rRNA synthesis during frog hibernation results in transformation of part of nucleoli in micronucleoli.     

Polyteny and endomitosis in supergiant trophoblast cells of the gray vole Microtus subarvalis

A cytomorphological study was made of peculiarly structured polytene chromosomes in supergiant trophoblast cells of Microtus subarvalis. The polyteny level was extremely high (over 1024C). The polytene chromosomes are characterized by a rather high degree of condensation of single chromosomes, and, as a consequence, close chromosome junctions and the typical disk pattern are lacking. The presence of complex nucleoli in the nuclei of these cells also testifies to a great detachment of chromonemes in polytene chromosomes of the studied supergiant trophoblast cells. Compared to other rodent species, a lower degree of chromoneme junction in the vole polytene chromosomes may cause their easy dissociation into single chromonemata, whose further condensation results in endomitotic chromosome formation. The chromosome depolytenization, earlier suggested from the analysis of interphase nucleus markers, has been traced here in detail. The process of polytene chromosome splitting was most obvious in the nucleolus-organizing chromosomes. A hony-combed nucleolus splits into numerous micronucleoli. The nucleus pattern becomes altered. Once in the polytene nucleus, chromosome bundles were located below the nuclear membrane and the central zone of the karyoplasm was not completely filled up. However, after dissociation of polytene chromosomes the whole karyoplasm was filled up with small nucleoli, and a thin layer of endomitotic chromosomes was seen beneath the nuclear membrane. The correlation between endomitosis and polyteny is discussed in terms of the dissociation of polytene chromosomes and formation of endomitotic chromosomes.     

Nucleoli in the cells of the erythrocyte and granulocyte series in the albino rat

In albino rats, as in other mammals, nucleoli with nucleolonemata and compact nucleoli in the stem cells of the erythrocyte and granulocyte series are progressively replaced during maturation and differentiation first by ring shaped nucleoli and then by micronucleoli with low and finally with inhibited RNA synthesis. There are, however, differences between the nucleolar coefficient values and the proportion of ring shaped nucleoli and micronucleoli in maturing neutrophils in the albino rat, the mouse and man. In the case of the erythroblasts, the differences between the proportion of various nucleolar types in the three given species are smaller. The results thus indicate that the developmental trend of nucleolar changes related to cell maturation and differentiation is the same, but that there are interspecies differences because of which the results obtained in one species cannot be applied mechanically to another, i.e. results obtained in a laboratory animal cannot be applied automatically to man.     

Some Chemical Properties of Isolated Pea Nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

Some chemical properties of isolated pea nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

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.     

Further studies of the nucleolar material in salivary gland nuclei of Sciara coprophila

The nucleolar-like bodies or micronucleoli of Sciara coprophila salivary gland nuclei have been studied with phase contrast, the Nomarski optics, Azure B staining, and electron microscopy. In the late fourth instar the main nucleolus formed at the X-chromosome may become extensively fragmented and may appear as a large aggregate of micronucleoli. At about the same time large numbers of micronucleoli in a more peripheral location are also found. Studies in partially squashed and stained nuclei, as well as in unfixed glands have shown that, at a time when the nucleolar material is abundant, the X-NOR is highly ramified with its branches permeating much of the nuclear space. These observations make it appear probable that most or all of the nucleolar material, even the more peripherally located, is actually in contact with the main nucleolar organizer or its branches. On the other hand, many chromosomal bands are also in close association with micronucleoli. At the level of electron microscopy some of the associations between chromo somal bands and micronucleoli are very intimate with the nucleolar material often found deep within the band. In other instances there seems to be physical continuity between extensions of band chromatin and certain areas of the fibrillar component. The bands in question could be the sites of secondary nucleolar organizers. In the electron microscope a large aggregate of micronucleoli, interspersed with portions of chromatin can often be seen in an approximately central location. This is interpreted as the main nucleolus with portions of its NOR. Both the main nucleolus and the more peripheral micronucleoli are indistinguishable in their fine structure and show the components typically found in nucleoli, i.e., fibrils and granules. On the other hand the fine structure of both RNA and DNA puffs is strikingly different.Supported by funds from Public Service Grants GM 12191 from the National Institute of General Medical Sciences and 5 RO 1 AM 10016-06 from the National Institute of Arthritis and Metabolic Diseases (to Dr. A. M. Garcia).     

Nuclear structures in the ovarioles of the butterfly Laspeyresia pomonella. Sex chromatin in trophocytes

The nuclear structures in the ovarioles have been studied in Laspeyresia pomonella by means of light and electron microscopy, autoradiography (RNA and DNA synthesis) and molecular hybridization in situ. The karyosphere was shown to form in oocyte nuclei at the beginning of oocyte growth. Numerous protein granules appeared in close contact with the karyosphere chromosomes; the true nucleolus was absent and the whole nucleus was inactive in RNA synthesis. A special attention was paid to studying nuclear structures in trophocytes. Numerous complex nucleoli actively synthesizing RNA formed in highly endopolyploid nuclei of trophocytes. Besides, each trophocyte had a spheroid vacuolized body of DNA which developed from one of meiotic bivalents soon after trophocyte differentiation and increased in diameter up to 10-15 mu. The DNA body in trophocytes and follicle cells was in close contact with the nucleolar material. Ribosomal DNA was present in these bodies as was shown by molecular hybridization in situ. A suggestion is put forward to the effect that the DNA bodies take part in the formation of complex nucleolar apparatus of trophocytes. On the basis of both the author's and literary data, a conclusion is drawn that DNA spheres in trophocytes and follicle cells are sex chromatin bodies formed, however, by both the X- and Y-chromosomes, rather than by one Y-chromosome.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

A STUDY OF THE NUCLEOLAR MATERIAL IN SCIARA COPROPHILA

In the polytene chromosomes of Sciara coprophila, in addition to a nucleolus, large numbers of nucleolarlike structures or micronucleoli are formed. A detailed mapping localized the nucleolar organizer at one end of the X chromosome and revealed that approximately 18% of the bands of each chromosome are potentially capable of producing micronucleoli. Most of these sites are in regions known from a previous study to show asynchronous DNA replication: DNA puffs and certain heterochromatic regions. Micronucleoli are rarely found in association with bulbs. The RNA metabolism of the polytene chromosomes during late fourth instar was studied using radioautographic techniques. Isolated glands were incubated in tritiated uridine for 10 to 30 min, and radioautographs were made of squash preparations. Despite the wide range of variation found among different larval cultures, the following pattern was observed. Just prior to and at the beginning of DNA puff formation, a period of intense extrachromosomal nucleolar and micronucleolar RNA synthesis occurs. After maximal development of the DNA puffs, the synthesis of extrachromosomal RNA is at a low point, while incorporation into bulbs and DNA puffs remains high. With the onset of the prepupal stage, all nuclear RNA synthesis ceases.     

The effect of the Bacillus thuringiensis exotoxin on the fine nucleolar morphology and ultrastructure

Depending on the dose administred to the experimental mice, the Bacillus thuringiensis exotoxin produces striking changes in the nucleolar morphology of hepatocytes such as the formation of ring-shaped nucleoli, micronucleoli and the segregation of nucleolar components. Such changes are apparently related to the decrease and inhibition of nucleolar biosynthetic activities in the production of the nucleolar RNA. In addition, the Bacillus thuringiensis exotoxin causes the formation of nucleolar peripheral dense plaques and, at higher concentrations, the segregation of two distinctly separated granular areas in the nucleolus. Both these light and dense granular areas showed positive staining with Bernhard's EDTA procedure for the preferential demonstration of RNA-containing structures. In some segregated nucleoli the granular components of light granular areas seemed to leave the nucleolus. The presence of discontinuous filamentous shell around micronucleoli produced by the high dose of exotoxin suggests the nucleolar origin of nuclear granular bodies which are surrounded by similar but continuous filamentous shell characteristic of these structures.     

Silver staining in transcriptionally active NORs of meiotic and mitotic cells in Acheta domesticus (L.) (Orthoptera)

The behaviour of the NOR material in mitotic and meiotic cells of Acheta domesticus was studied by silver staining. — In mitotic chromosomes black silver staining is observed in the centromeric region of 2 pairs of acrocentric chromosomes. Additionally a polymorphic silver positive region is found at the telomere of a large submetacentric chromosome. — The Ag-pattern of the amplified rDNA material in various stages of oogenesis was followed. During pachytene the extra DNA body shows dark brownish staining and only a few black spots. One distinct black precipitate, however, is found in association with meiotic chromosomes. In early diplotene the central core of the extra DNA body is heavily stained with silver. The outer shell shows only brown staining. In the following stages of diplotene the compact structure of the outer shell is loosened and small brown extra nucleoli are found in the remaining nucleus. These nucleoli show black Ag-precipitates in their centres. During the desintegration of the extra DNA body the nucleus becomes filled with small extra nucleoli. The black stained central core is reduced in size and finally disappears.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.