Frequency of Ag-stained nucleolus organizer regions in the acrocentric chromosomes of man

Summary The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding of cultured lymphocytes in 51 karyotypically normal persons (31 males and 20 females). A consistent pattern of Ag-positive NORs was found in each individual. Ninety percent of the individuals have a modal number of 8–10 Ag-positive NORs per cell. The frequency of Ag-positive NORs is similar in all five acrocentrics. A statistically nonsignificant lower frequency is found in chromosome 22. Ag-negative NORs on both homologues were found in four cases. The observed frequency distribution of individuals with homozygous NOR-positive, heterozygous, and homozygous negative acrocentric chromosomes was in accordance with the Hardy-Weinberg law in all five pairs of the acrocentric chromosomes as well as in total. No sex difference was observed in our material.A.-V. Mikelsaar is visiting exchange scientist of the Österreichische Bundesministerium für Wissenschaft und Forschung     

Population polymorphism of silver-stained nucleolar organizer chromosome regions in man

Activity of nucleolar organizer regions (NORs) of acrocentric chromosomes determined by Ag-staining was comparatively studied in 40 individuals of Bulgarian and 40 individuals of Russian populations. Chromosome 21 was found to be significantly more often stained in both populations. The other NORs did not differ significantly in staining from the means. No differences were noted between individual NORs, in respect of the intensity of Ag-staining in both populations, except chromosome 15 which showed markedly decreased staining capacity in Russians. The data obtained are compared with those published in literature concerning four other populations.     

Argyrophilic nucleolar organizer regions

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining, may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.     

Frequency of Ag-stained nucleolus organizer regions in the chromosomes of cattle

Sequential Ag NOR/G-band studies were performed on the chromosomes of 33 Austrian Al bulls. The mean number of Ag-NORs per bull karyotype was 6.06. The distribution of Ag-NORs was found to be characteristic for each individual.     

Importance of interphase nucleolar organizer regions in tumor pathology

The importance of the distribution of silverstained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.     

Polymorphism of nucleolar organizer regions of chromosomes in human embryos

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.     

Importance of interphase nucleolar organizer regions in tumor pathology.

The importance of the distribution of silver-stained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.     

Polymorphism of Ag-stained nucleolus organizer regions in lymphocytes of patients with ovarian or breast adenocarcinoma

Summary The Ag-stainability of nucleolus organizer regions (NORs) in the acrocentric chromosomes identified by Q-banding was studied in 45 female patients with adenocarcinoma of the ovary or breast and in 45 healthy females. Significantly higher frequencies of Ag(+)NORs per individual (8.8 and 8.3; P<0.05), in the G group chromosomes (3.6 and 3.2; P<0.05), and in chromosome 21 (1.9 and 1.7; P<0.02) were found in patients, compared with controls. Despite the lack of significant differences in NORs between the groups of patients with ovarian and breast adenocarcinoma, the main difference between the patients and controls was due to the patients with adenocarcinoma of the ovary, where a significantly higher frequency of Ag(+)NORs was found in chromosomes 21 (P<0.01) and 13 (P<0.05).     

Dimorphic nucleolar organizer regions in the frog Rana blairi.

The nucleolar organizer-specific staining procedure, ammoniacal silver (Ag-AS), has been used to study the distribution and size of the nucleolar organizer regions (NORs) in chromosomes of the frog Rana blairi (Mecham, Littlejohn, Oldham, Brown and Brown). The somatic metaphase karyotype of this frog is similar to that of other frogs of the Rana pipiens species complex, numerically (2n=26) and morphologically. Secondary constrictions are detectable in untreated Giemsa-stained metaphase preparations as achromatic gaps in the long arms of a pair of submetacentric chromosomes (no. 10). These constrictions are the only regions which are deeply stained with the Ag-AS method and are thus identified as the nucleolar organizer regions (Ag-NORs). In each of the three individuals, the Ag-NORs as visualized on the homologues are of unequal length.     

Compensatory changes in silver-stainability of nucleolar organizer regions in mice.

Silver-stainability of nucleolar organizer regions (NORs) that contain genes for ribosomal RNA (rDNA) was investigated using two mouse strains, BALB/cCrSlc and MOA, and their hybrid progeny. The patterns of segregation of the rDNA clusters were analyzed in terms of chromosomal C-banding and by use of a polymorphic probe for the variable region in backcrossed N2 and N3 individuals. The results indicate that the intensity of Ag-NOR staining is stably inherited in most of the rDNA clusters, irrespective of different genetic backgrounds. In some clusters, such as those on chromosome 12 of BALB/cCrSlc, a modulation of the intensity is observed. This modulation seems to be due to compensatory activation via a change in the number of actively transcribed genes. The change from silver-negative to silver-positive staining of the NOR of chromosome 12 of BALB/cCrSlc was correlated with demethylation of the genes.     

Population heteromorphisms of Ag-stained nucleolus organizer regions (NORs) in the acrocentric chromosomes of East Indians

Summary The nucleolar organizer regions (NORs) of acrocentric chromosomes in 70 normal East Indians were examined by Ag-staining (NSG) and acridine orange reverse banding (RFA) techniques. The Ag-stainability of NORs was variable from one individual to another but characteristics were constant within each individual. The average modal number of Agpositive NORs per individual was eight. A racial difference in the expression of NORs is suggested. to study the heteromorphism of NORs, the NSG technique was found to be more useful than RFA. Furthermore, it is concluded that there is no direct relationship between a heteromorphism of NORs identified by NSG and that identified by the RFA technique. Quantitative data on these differences is provided. In addition NOR-regions are classified into five sizes namely; very large, large, medium, small, and very small using subjectively defined criteria.     

Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining

A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.     

Chromosome banding in salmonid fishes: nucleolar organizer regions in Oncorhynchus

Chromosome banding patterns obtained by silver staining and chromomycin a3 (CMA3) staining were analyzed in six species of Oncorhynchus: O. tshawytscha, O. kisutch, O. keta, O. nerka, and O. gorbuscha from North America and O. masou from Japan. Four different chromosomal locations of the nucleolar organizer regions (NORs) were found in different species. In O. tshawytscha, O. kisutch, and O. masou the NORs comprised the entire short arms of one medium-sized acrocentric chromosome pair. In O. nerka the NORs were found in an interstitial band on the short arms of one submetacentric chromosome pair and in O. gorbuscha proximal to the centromere on one metacentric chromosome pair. In O. keta the NORs were found on the telomeres of one small submetacentric chromosome pair. As in the related genera Salmo and Salvelinus chromomycin A3 positive bands were found at the same sites as the AgNORs in all species. Salmonid fish are assumed to be ancestral tetraploids and the considerable differences in chromosome number between different species are thought to be the result of chromosomal fusions after tetraploidization. In all members of the genus Oncorhynchus the rearrangements have resulted in the consolidation of the NORs on a single chromosome pair. The possible significance of intra- and inter-species NOR polymorphisms is discussed.     

Quantitative analysis of the variability of nucleolar organizer regions in Salmo trutta

A quantitative analysis combined with Ag staining was carried out to study the size variation of the main nucleolar organizer region (NOR) bearing chromosome pair 11 of Salmo trutta. A standardized NOR size measurement was developed by comparing the length of the short arm (NOR-bearing region) to the total chromosome length. Statistical procedures support arguments for the existence of a large and structural polymorphism within this species for this chromosome region. A minimum of five different chromosome classes were identified, which account for the total variation found. Size variation among classes was due both to changes in the number of NOR clusters as well as to the amount of rDNA genes within each cluster. NOR size values were normally distributed in the sample analyzed.     

Prognostic value of nucleolar organizer regions in neuroendocrine tumours of the lung

The value of the number and size of the nucleolar organizer regions (NORs) as prognostic indicators in human neuroendocrine lung tumours was evaluated in a quantitative study of 57 cases, including 33 small cell carcinomas (SCLCs), 9 well-differentiated neuroendocrine carcinomas (WDNECs) and 15 classic carcinoids. NORs were visualized on paraffin sections by an argyrophilic technique (AgNOR) and measured by automatic image analysis. In each case, the mean number and area of AgNORs were evaluated; the results were compared with clinical follow-up and survival. AgNOR values for both number and area were significantly higher in SCLCs than in WDNECs and carcinoids. WDNECs had insignificantly higher AgNOR values than carcinoids. Among SCLCs, AgNOR values of the oat cell subtype and the intermediate cell subtype did not differ significantly. Regardless of the histological tumour type, AgNOR values strongly correlated with prognosis, with more and larger AgNORs indicating a more progressive clinical course. In the present study we demonstrate for the first time that the biological behaviour of neuroendocrine lung tumours is correlated with the number and size of AgNORs. Thus the measurement of AgNORs may serve as an additional prognostic indicator in these neoplasms, particularly in the separation of SCLCs from WDNECs with a more favourable prognosis.     

Expression of nucleolar organizer regions (NORs) in ovarian epithelial tumors

AgNOR staining technique was tested in ovarian epithelial tumors to evaluate its diagnostic potential in distinguishing between borderline tumors and well-differentiated carcinomas. In our opinion, the AgNOR count appears useful for assessing differences only between borderline and well-differentiated serous ovarian tumors at stage I of FIGO clinical advancement.     

Non-nucleosomal configuration of chromatin in nucleolar organizer regions of metaphase chromosomes in situ

The structure of metaphase chromatin in a human tumor cell line, TG cells, was investigated using thin sections selectively stained for DNA with the Feulgen-like osmium-ammine reaction. The bulk of metaphase chromatin was characterized by the nucleosomal configuration. Some specimens were pretreated by silver staining for selective visualization of acidic proteins of the nucleolar organizer regions. In these specimens, the osmium-amine DNA tracer revealed that the chromatin present at the sites of silver granule localization had a completely extended configuration, and never gave rise to nucleosomal structures.     

Chromosome banding in salmonid fish: nucleolar organizer regions in Salmo and Salvelinus

Chromosome banding patterns obtained by silver staining (Ag-NORs) were analyzed in three species of Salmo (rainbow, brown trout, and Atlantic salmon) and three species of Salvelinus (brook trout, lake trout, and arctic char). In rainbow trout and Atlantic salmon the Ag-NORs were found at the secondary constrictions of a single chromosome pair, while in brown trout the Ag-NORs were found on the short arms of one or two of the two longest subtelocentric or acrocentric chromosome pairs. The location of the Ag-NORs was multichromosomal in the three Salvelinus species, occurring on one or both members of four to six different chromosome pairs in different individuals. The Ag-NOR sites were on the short arms of some acrocentric pairs and at the telomeres of other acrocentric pairs and one or two metacentric pairs. Chromomycin A3 positive bands were found at the same sites as the Ag-NORs in all species. In the species with multichromosomal location of Ag-NORs, polymorphisms in the size and location of the NORs were extremely common, so that almost every individual fish had a different pattern of Ag-NOR sites.