Nucleolar changes in human phytohaemagglutinin-stimulated lymphocytes

Summary The nucleoli of lymphocytes from circulating peripheral blood and from phytohaemagglutinin (PHA)-stimulated cultures (from 2 h–96 h) were studied using a silver method, RNA-specific fluorescent staining, and electron microscopy of ultrathin sections. In peripheral blood about 75% of the lymphocytes have one ring-shaped nucleolus composed of a distinct fibrillar centre surrounded by a dense pars fibrillaris and little granular material; the remaining lymphocytes showing two or more small ring-shaped nucleoli. With PHA stimulation, the number of cells with several nucleoli increases first (from 2 h–12 h). Next, cells containing one or, at most, two large nucleoli with nucleolonema devoid of fibrillar centers are seen (from 4 h on). 34 h after PHA, nucleoli of the compact type containing one or more fibrillar centres appear and comprise about 60% of the cells after 72 h. The appearance of more than one nucleolus per cell shortly after PHA administration suggests an activation of additional nucleolar organizer regions (NOR), which fuse to form one or two large nucleoli with nucleolonema. These are then transformed into compact nucleoli. The fibrillar centers stain preferentially with silver. They contain nonchromosomal proteins and may serve as stores for nucleolar proteins. The fusion of activated NORs during the first cell cycle explains the relatively high frequency of satellite associations in first mitoses compared to later mitoses after stimulation.     

Analysis of ring-shaped nucleoli in serially sectioned human lymphocytes

Summary Serial section analysis has demonstrated that ring-shaped nucleoli of mature human lymphocytes are spherical structures consisting of a peripheral ribonucleoprotein shell that surrounds one large fibrillar center. The shell exhibits usually one or, less frequently, two openings. The fibrillar center is in contact with the nucleoplasm and perinucleolar condensed chromatin, which frequently appears as a pedicle-like structure. Several chromocenters are associated with the ring-shaped nucleolus.     

Intercellular NOR-Ag-variability in man. II. Search for determining factors,clonal analysis

Summary Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts.In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.     

Studies on satellite nucleoli in human normal and leukemic lymphocytes

Lymphocytes from normal and leukemic patients, and phytohemagglutinin-stimulated lymphocytes were investigated by means of immunofluorescence procedures and a silver reaction for the demonstration of proteins characteristic of nucleolus organizer regions in interphasic cells to provide basic information on the presence of satellite nucleoli in these cells. The results clearly indicated that satellite nucleoli are present in a limited but constant percentage of peripheral lymphocytes. An increased percentage of lymphocytes with satellite nucleoli was found only in leukemic patients and after silver staining. In contrast, a decreased percentage of satellite nucleoli was found 24 h after stimulation with phytohemagglutinin vitro. In leukemic patients, the discrepancy in the percentage of lymphocytes with satellite nucleoli between immunostained and silver-stained preparations may suggest that the silver reaction demonstrates the presence of an additional argyrophilic protein besides proteins B23 and C23, or altered forms of these proteins, which does not react with the specific antibodies.     

Ribosomal RNA expression in a mammalian hybrid,the hinny

The expression of nucleolus organizer region (NOR) activity in diploid cells was investigated in a model mammalian hybrid system, the hinny (female ass x male horse), by sequential Ag-NOR and chromomycin A3/distamycin A/DAPI (CDD) staining in lectin-stimulated peripheral blood lymphocytes. In the majority of cases we found non-expression of the horse-derived NOR chromosomes in the hinny. However, in one case there was strong NOR expression on horse-derived chromosome no. 1.     

Electron microscopic study of Ag-protein localization in the nucleoli of human lymphocytes

The present study was undertaken to provide more information on the ultrastructural localization of a silver reaction in normal resting and stimulated lymphocytes as well as leukaemic resting lymphocytes. The results obtained indicated that in the ring-shaped nucleoli of normal mature lymphocytes silver stained proteins (SSPs) were present mostly within single fibrillar centers. In the nucleoli of lymphocyte cultures, being in the presence of phytohemagglutinin (PHA) for 6--72 hours, SSPs formed finger or loop-like protrusions from fibrillar centers towards the adjacent areas of the nucleoli. In the ring-shaped nucleoli of mature leukaemic lymphocytes SSPs are present not only within fibrillar centers, but also in protrusions diverging from fibrillar centers into the surrounding peripheral nucleolar ring. In this respect the nucleoli of leukaemic mature lymphocytes were similar to normal lymphocytes shortly after mitogen stimulation.     

Position-dependent NOR activity in barley

Two types of intraspecific nucleolar dominance/suppression are described for barley,Hordeum vulgare L. When the nucleolus organizing regions (NORs) originally belonging to chromosomes 6 and 7 are combined by translocation in one chromosome, NOR 6 is dominant over NOR 7. Neither significant loss of rDNA nor its hypermethylation is the reason for the reduced nucleolus forming activity of NOR 7. Intrachromosomal NOR suppression probably does not occur in isochromosome 6s, which has two NORs 6 in one chromosome. Meiotic and somatic pairing of the homologous arms might be the reason for early fusion of their nucleoli and thus for the lower than expected maximum number of interphase nucleoli. Variable suppression of a partial NOR (6³) is described for descendants of crosses between translocation lines with split NORs 6 and 7. In these cases also, the reduced activity of the partial NOR 6³ is not due to deletion of rDNA as shown by in situ hybridization. Unstable methylation of NOR 6³ in heterozygous F₁ individuals is probably the cause of this phenomenon.     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.     

The behavior of nucleolus organizers in structurally changed karyotypes of barley

Two standard karyotype barley lines and 18 lines with karyotypes reconstructed by means of induced reciprocal translocations have been studied with respect to nucleolus formation. The standard karyotype contains two pairs of satellite chromosomes (pairs 6 and 7). Five of the structurally changed karyotypes contain, as a result of reciprocal translocations between the standard satellite chromosomes, only one satellite chromosome pair, each chromosome with two satellites and two nucleolus organizing regions. Under these circumstances, only two of the four NORs are active in nucleolus formation while the other two — probably the transposed ones — remain inactive; hence the maximum number of primary nucleoli per nucleus is two. — When NORs are translocated to chromosomes with no NOR in the standard karyotyp, the normal pattern of nucleolus formation remains unchanged. The same is true after transposition of segments from other chromosomes to the satellites of the standard SAT-chromosome pairs 6 and 7. The results obtained are discussed with respect to effects of translocations on the activity and behaviour of nucleolus organizing regions.     

Ultrastructure and functions of ring-shaped nucleoli, fibrillar centers and nucleolar vacuoles

Evidence on the ultrastructure of ring-shaped nucleoli in several cell types is reviewed. Detailed attention is paid to these structures in PEK culture cells after the action of actinomycin D, to infiltrating carcinomas of human breast after different degrees of malignization, and to hepatocytes of 11 day old chick embryos. On the basis of our own and literary data, the ring-shaped nucleoli are classified into two groups: 1) those with centrally located fibrillar centres, and 2) those containing large central vacuole. Connections of fibrillar centres with the nucleolus organizer regions in the interphase nucleolus and mitotic chromosomes are considered in detail. Literary and our own data on the genesis and functional activity of ring-shaped nucleoli with the central vacuole, as well as nucleoli with different degrees of vacuolization (vacuolized nucleoli) are specially analyzed. Cellular function, properties and significance of the central vacuole in low differentiated cells are discussed.     

Further studies on satellite nucleoli in rat and mouse hepatocytes

To provide more information on satellite nucleoli, these nuclear structures were studied by means of cytochemical and immunofluorescence procedures in rat and mouse hepatocytes without and following experimental inhibition of the RNA synthesis. The immuno-staining specific for nucleoli or B23 as well as C23 proteins demonstrated that satellite nucleoli and characteristic nucleoli exhibit the same fluorescence. The number of satellite nucleoli decreased after inhibition of nucleolar RNA synthesis in a similar way to the number of silver-stained granules (SSGs) of characteristic nucleoli. Inhibition of RNA synthesis also reduced the number of hepatocytes containing satellite nucleoli. Thus, satellite nucleoli seem to be real nucleoli from single NORs which did not fuse in the formation of a characteristic nucleolus.     

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.     

Distribution of fibrillar centers and silver-stained components in the nucleolus of human Sertoli cells

The nucleolus of the human Sertoli cell consistently showed three distinct, spontaneously segregated parts: 1. one or two large, silver-positive fibrillar centers; 2. strands of dense fibrillar component continuous with the dense cords surrounding the fibrillar center. These components were also silver-positive; 3. a granular, silver-negative mass. These observations show that in the human Sertoli cell the number of fibrillar centers is far lower than the diploid number of NORs. They also suggest that the fibrillar center might contain several NORs in this cell type.     

Genetic studies on nucleolus organizer regions (NORs) in cattle

Experimentally produced monozygotic twins, natural opposite sex blood chimeras (freemartins), and several pedigrees were used to evaluate the genetic influences on the nucleolus organizer region (NOR) patterns in cattle. In monozygotic twins, the NOR patterns of both twins are extremely similar. In chimeras, NOR patterns of genetically identical, peripheral blood lymphocytes (PBL) from the two partners resemble each other. In contrast, genetically different PBL (sib organ) differ significantly in the same environment. A high heritability of the individual NOR patterns is also demonstrated in our 23 pedigrees. In conclusion, our data demonstrate that variation in NOR expression is predominantly due to genetic factors.     

Tissue and sex differences in the expression of nucleoli in mouse somatic cells.

In Mus musculus, the nucleolus organizer regions (NORs), or sites of ribosomal RNA-encoding genes, map at three chromosomal pairs. A silver procedure was modified to stain nucleoli in interphasic somatic cells of mice. The number of nucleoli per cell nucleus was determined in squashed cells of kidney, liver and pancreas obtained from male and female mice. In liver and pancreas cells the average number of nucleoli per cell was 4.84 and 4.66, respectively, and only 2.83 in kidney cells (p < 0.001). Less than 8% of pancreas cells and about 15% of liver cells contained more than 6 nucleoli per cell, which was the maximum expected number. In addition, the number of nucleoli per cell was significatively different (p < 0.01) when male and female liver or pancreas cells (not kidney cells) were compared. In both cases, female cells presented more nucleoli than the respective male cells. Assuming that the available NORs are the same, the variable number of nucleoli in the examined cell types would be the consequence of a tissue specific NOR regulation. The apparent influence of sex on this regulation is noted.     

A chromosome rearrangement in Neurospora that produces segmental aneuploid progeny containing only part of the nucleolus organizer

In translocation T(ILVL)OY321 of Neurospora crassa a distal portion of the nucleolus organizer chromosome, including ribosomal DNA sequences and the nucleolus satellite, is interchanged with a long terminal segment of IL. When OY321 is crossed by Normal sequence, one-fourth of the meiotic products are segmental aneuploids that contain two copies of the long IL segment and that are deficient for the distal portion of the organizer. Each such product forms a nucleolus and is viable. The complementary aneuploid products are deficient for the IL segment and are therefore inviable. — In crosses of OY321xOY321, each product is capable of making two nucleoli; nucleoli formed by the separated nucleolus organizer parts usually fuse, but most 8-spored asci contain some nuclei in which two separate nucleoli can be seen. One nucleolus is then terminal on its chromosome while the second is interstitial and somewhat smaller. — In crosses of OY321 x Normal, half of the meiotic products are capable of making two nucleoli. However, only about 15% of 8-spored asci have one or more nuclei containing separate nucleoli. At pachytene and later in prophase I, the single fusion nucleolus is associated with three bivalent chromosome segments. Each nucleus of every ascus contains at least one nucleolus, even in asci where some nuclei display two nucleoli. — Crosses of Aneuploid x Normal are usually semibarren, producing a reduced number of ascospores, some of which are inviable. Some aneuploid cultures become fully fertile by reverting to a quasinormal sequence lacking a satellite. In some crosses of Aneuploid x Normal, individual asci may show at prophase I either complete loss, partial loss, or pycnosis of the translocated IL segment. This observation of pycnosis suggests chromosome inactivation. — Growth from aneuploid ascospores is initially slow, but can accelerate to the wild-type rate.