Nucleolar Organizer Research Articles

Epigenetic Changes of Activity of the Ribosomal Cistrons of Human Acrocentric Chromatids in Fetuses, Middle-aged (22–45 years) and Old Individuals (80–106 years)

The level of total heterochromatin, Ag-positive nucleolar organiser regions (NORs) heterochromatin, non-associated and associated heterochromatin satellite stalks of acrocentric chromatids (some acrocentric chromosomal chromatid satellite stalks are connected to each other forming a satellite association), the intensity of each acrocentric chromatid involved in the association and types of associated chromatids were studied from 29 fetuses, from 32 healthy individuals 22–45 years (middle-aged) old and 22 healthy individuals 80–106 years old. The chromosomes were identified by the analysis of G-banding, using the Ikaros karyotyping system (Meta system). The differential scanning calorimeter showed an increase in chromatin thermostability (heterochromatinization) at adults (middle and old aged) compared with fetuses. The number of Ag-positive NORs per cell, for both associated and non-associated chromatids, was significantly increased in fetus cells than in middle and in extreme old age. The chromatid satellite association of fetus cells and the elderly was consistently reduced, compared with middle-aged individuals. Activity of chromatids of the 15th chromosomes, entered in association in the fetus, in middle and old age adults, was significantly reduced (p < 0.05), compared to other acrocentric chromatids, while the chromatids of 21st chromosome participated in associations with high activity (p < 0.05). The frequency of association of homologous (13:13; 14:14; 15:15; 22:22) and certain types of non-homologous chromatids (15:22 and 21:22) in the fetus and in 22–45 and 80–106 years old individuals was almost identically. The above noted phenomena apparently indicate that chromatid satellite ribosomal genes of acrocentric chromosomes undergo specific epigenetic variation depending on age, determining the specific synthesis of rRNA for constructing specific ribosomes, which may have great importance in assessing the general functioning of cells in normal and pathological conditions.

NORs on human acrocentric chromosome p-arms are active by default and can associate with nucleoli independently of rDNA

Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.

Diagnostic and Prognostic Significance of AgNOR Counts in Before and After Radiotherapy-Treated Cervical Carcinogenesis is Compared with Cervical Cytology and Histology

Nucleolar organizer region-associated proteins are argyrophilic and visualized by silver stains. There are two types of dots are present. One is the presence of single dots, and another is pleomorphic dots. Pleomorphic dots are few, increase with severity of cervical lesions and hence indicate a more severe prognosis. The present study aimed at investigating the diagnostic value of both pleomorphic and single AgNOR dots in cervical carcinogenesis in different cytological stages of cervix before and after radiotherapy. The results were correlated with cervical cytology and histopathology. The study was conducted on the samples of 37 untreated cases of patients coming to the outpatient door of AHRCC. In two slides, samples were taken for PAP smear cytology and AgNOR staining. From histopathological detection, various grades of squamous cell carcinoma of the cervix were determined. Samples were divided into cytological/histological normal/controls (N = 10), inflammation (N = 7), LSIL (N = 8), HSIL (N = 7) and invasive carcinoma (N = 5). AgNOR staining was performed slides containing cervical smears. Samples from same patients [LSIL (N = 8), HSIL (N = 7) and invasive carcinoma (N = 5)] that were subjected to radiotherapy were assessed again for AgNOR scoring. The rate of increase in cell counts of pleomorphic dots was higher than the rate of increase in cell counts of single dots, and there is a positive and significant correlation of cell counts of both pleomorphic and single dots with disease severity. The results were correlated with PAP stained cervical cytology and histopathology to find out the diagnostic and prognostic significance. In squamous cells of the normal/control group, the AgNORs were fine and centrally placed in the nucleoli. AgNORs in the case of LSIL, HSIL and invasive carcinoma of the cervix before radiotherapy were large and variable in size and shape. The cases studied for AgNORs count after 6 month of radiotherapy showed less number of AgNORs dots which were single and fewer in numbers as compared to the pre-radiation group. AgNOR pleomorphic dot counts increase with severity of cervical lesions and may be used as an effective tool along with cervical cytology and histopathology for detection of cervical carcinogenesis.

Double Robertsonian translocations in an infertile patient with macrocytic anemia: a case report

BackgroundConstitutional heterologous double Robertsonian translocations (DRT) between chromosomes 13/14 and chromosomes 14/15 with 44 chromosomes are extremely rare. In this case report, we present the karyotype analysis of metaphases prepared from bone marrow, peripheral blood and cultured skin tissue cells. These showed only 44 chromosomes with DRT involving chromosomes 13, 14 and 15. To our knowledge this is the first reported case with DRT involving chromosomes 14 and 15.Case presentationThe patient is an 81-year-old infertile male with a history of persistent macrocytic anemia (MA). The patient presented with fatigue, paleness of the skin, shortness of breath, lightheadedness and occasional dizziness. Work-up for common causes of macrocytic anemias in this case were excluded: folate/vitamin B12 deficiency, hypothyroidism, liver diseases, hemolysis, bleeding, alcoholism, exposure, HIV infection, chemotherapy or blood loss, drug-toxicity effect, or myelodysplasia. This individual with DRT had only six nucleolus organizer regions (NORs), instead of the usual ten, of which 50% of the 6 NORs were inactive (n = 3).ConclusionIn this case, macrocytic anemia (MA) appeared to be due to reduction in active NORs in DRT. We postulate that the marked reduction in active NORs leads to reduction in active nucleoli formation, which may be limiting ribosomal RNA synthesis, contributing to MA. It is probable that reduction in NOR activity affected normal DNA synthesis and cellular functions.

Chromosome Dynamics Regulating Genomic Dispersion and Alteration of Nucleolus Organizer Regions (NORs).

The nucleolus organizer regions (NORs) demonstrate differences in genomic dispersion and transcriptional activity among all organisms. I postulate that such differences stem from distinct genomic structures and their interactions from chromosome observations using fluorescence in situ hybridization and silver nitrate staining methods. Examples in primates and Australian bulldog ants indicate that chromosomal features indeed play a significant role in determining the properties of NORs. In primates, rDNA arrays that are located on the short arm of acrocentrics frequently form reciprocal associations (“affinity”), but they lack such associations (“non-affinity”) with other repeat arrays—a binary molecular effect. These “rules” of affinity vs. non-affinity are extrapolated from the chromosomal configurations of meiotic prophase. In bulldog ants, genomic dispersions of rDNA loci expand much more widely following an increase in the number of acrocentric chromosomes formed by centric fission. Affinity appears to be a significantly greater force: associations likely form among rDNA and heterochromatin arrays of acrocentrics—thus, more acrocentrics bring about more rDNA loci. The specific interactions among NOR-related genome structures remain unclear and require further investigation. Here, I propose that there are limited and non-limited genomic dispersion systems that result from genomic affinity rules, inducing specific chromosomal configurations that are related to NORs.

Nucleolar organizer regions in human oral verrucous carcinoma and adjacent lining epithelium

ABSTRACT The epithelium adjacent to an oral squamous cell carcinoma is at risk of undergoing precancerous changes. Even after such changes occur, however, the adjacent epithelium remains histologically similar to normal mucosa. We investigated five argyrophilic nucleolar organizer region (AgNOR)-related features in samples of oral verrucous carcinoma (VeCa) and their corresponding adjacent lining epithelium (adj. VeCa). Morphometric characteristics of AgNORs in oral adj. VeCa and oral VeCa were compared to normal mucosa epithelium, squamous cell carcinoma and oral mucosa epithelium adjacent to squamous cell carcinoma findings that we published earlier. Although adj. VeCa and normal oral mucosa were histologically similar, total AgNOR volume differentiated adj. VeCa from normal oral mucosa, but revealed no significant difference between VeCa and adj. VeCa. Total AgNOR volume/nuclear volume discriminated VeCa from adj. VeCa and normal oral mucosa. Certain AgNOR parameters provide a complementary tool for discriminating VeCa from adj. VeCa and normal oral mucosa, and also for detecting incipient malignant changes in epithelium adjacent to VeCa. Use of the AgNOR technique is cost-effective, because it can be performed on paraffin sections.

Comparative Cytogenetics of Two Squalius Bonaparte, 1837 Species (Cypriniformes: Leuciscidae)

The karyotypes and other chromosomal characteristics of Anatolian endemic species Squalius carinus Ozulug and Freyhof (Ichthyol Explor Freshw 22(2):107–148, 2011) and S. fellowesii (Gunther, 1868) (Cypriniformes: Leuciscidae) were analysed by means of sequential Giemsa staining, C-banding and silver staining. The diploid chromosome numbers were invariably 2n = 50; their karyotypes were composed of 12 pairs of metacentric, ten pairs of submetacentric and three pairs of subtelo-acrocentric chromosomes in S. carinus, whereas ten pairs of metacentric, ten pairs of submetacentric and five pairs of subtelo-acrocentric chromosomes in S. fellowesii. No heteromorphic sex chromosomes were detected. The largest chromosome pair in their karyotypes was a subtelo-acrocentric chromosome. C-positive heterochromatins were observed on the pericentromeric regions of most of the chromosomes in the studied species. In addition, heterochromatic blocs were observed in the karyotype of S. carinus. The nucleolus organizer regions were detected terminally on the short (p) arms of single submetacentric chromosome pair in both species. Our study thus confirmed overall conservatism of leuciscin karyotypes.

H3K9 Demethylation-Induced R-Loop Accumulation Is Linked to Disorganized Nucleoli

The nucleolar structure and integrity are important for a range of cellular functions of the nucleoli. It has been shown that cells lacking histone H3 Lysine 9 (H3K9) methylation form fragmented nucleoli. However, the molecular mechanism involved remains poorly understood. Here, we present evidence suggesting that loss of H3K9 dimethylation (H3K9me2) triggers R-loop accumulation at the rDNA locus, which further leads to the multilobed nucleoli. We reveal that suppression of H3K9 methyltransferase G9a by the inhibitor BIX 01294 causes R-loop accumulation at the rDNA region as well as inducing formation of multiple nucleoli. SiRNA-mediated knockdown of RNase H1 which can hydrolyze the RNA chain in R-loops causes an increase in R-loop formation, which in turn results in multiple nucleoli in one nucleus, whereas H3K9me2 levels are not affected by R-loop accumulation. Inhibition of RNA polymerase I transcription elongation by small molecule inhibitors induces a substantial decrease in H3K9me2 levels, accumulation of R-loops at rDNA sites, and nucleolus fragmentation. These results provide a mechanistic insight into the role of H3K9me2 in the structural integrity and organization of nucleoli via regulating R-loop accumulation.

Comparative cytogenetics of the ground frogs Eupsophus emiliopugini Formas, 1989 and E. vertebralis Grandison, 1961 (Alsodidae) with comments on their inter- and intraspecific chromosome differentiation.

South American frogs of the genus Eupsophus Fitzinger, 1843 comprise 10 species. Two of them, Eupsophus vertebralis Grandison, 1961 and E. emiliopugini Formas, 1989 belong to the Eupsophus vertebralis group, exhibiting 2n = 28. Fundamental number differences between these species have been described using conventional chromosome staining of few specimens from only two localities. Here, classical techniques (Giemsa, C-banding, CMA3/DAPI banding, and Ag-NOR staining), and fluorescence in situ hybridization (FISH, with telomeric and 28S ribosomal probes), were applied on individuals of both species collected from 15 localities. We corroborate differences in fundamental numbers (FN) between E. vertebralis and E. emiliopugini through Giemsa staining and C-banding (FN = 54 and 56, respectively). No interstitial fluorescent signals, but clearly stained telomeric regions were detected by FISH using telomeric probe over spreads from both species. FISH with 28S rDNA probes and Ag-NOR staining confirmed the active nucleolus organizer regions signal on pair 5 for both species. Nevertheless, one E. emiliopugini individual from the Puyehue locality exhibited 28S ribosomal signals on pairs 4 and 5. Interestingly, only one chromosome of each pair showed Ag-NOR positive signals, showing a nucleolar dominance pattern. Chromosomal rearrangements, rRNA gene dosage control, mobile NORs elements, and/or species hybridization process could be involved in this interpopulation chromosomal variation.

Interspecific cytogenetic relationships in three Acestrohynchus species (Acestrohynchinae, Characiformes) reveal the existence of possible cryptic species.

The karyotypes and chromosomal characteristics of three Acestrorhynchus Eigenmann et Kennedy, 1903 species were examined using conventional and molecular protocols. These species had invariably a diploid chromosome number 2n = 50. Acestrorhynchus falcatus (Block, 1794) and Acestrorhynchus falcirostris (Cuvier, 1819) had the karyotype composed of 16 metacentric (m) + 28 submetacentric (sm) + 6 subtelocentric (st) chromosomes while Acestrorhynchus microlepis (Schomburgk, 1841) had the karyotype composed of 14m+30sm+6st elements. In this species, differences of the conventional and molecular markers between the populations of Catalão Lake (AM) and of Apeu Stream (PA) were found. Thus the individuals of Pará (Apeu) were named Acestrorhynchus prope microlepis. The distribution of the constitutive heterochromatin blocks was species-specific, with C-positive bands in the centromeric and telomeric regions of a number of different chromosomes, as well as in interstitial sites and completely heterochromatic arms. The phenotypes of nucleolus organizer region (NOR) were simple, i. e. in a terminal position on the p arm of pair No. 23 except in A. microlepis, in which it was located on the q arm. Fluorescence in situ hybridization (FISH) revealed 18S rDNA sites on one chromosome pair in karyotype of A. falcirostris and A. prope microlepis (pair No. 23) and three pairs (Nos. 12, 23, 24) in A. falcatus and (Nos. 8, 23, 24) in A. microlepis; 5S rDNA sites were detected in one chromosome pair in all three species. The mapping of the telomeric sequences revealed terminal sequences in all the chromosomes, as well as the presence of interstitial telomeric sequences (ITSs) in a number of chromosome pairs. The cytogenetic data recorded in the present study indicate that A. prope microlepis may be an unnamed species.

Histopathological Classifications of Oral Leukoplakia and its Relation to Cell Proliferative Activity: A Case Series

This study relates the average number of nucleolar organizing regions (NORs) obtained in a series of cases of oral leukoplakia, with three methods of histopathological classification of such lesion. This is a histopathological-histochemical laboratory cross-sectional study. The 18 cases of leukoplakia analyzed were filed at Pathology Service of the Biological Sciences Institute of the University of Passo Fundo, Rio Grande do Sul, Brazil (SDH/ICB/UPF) (2017 and 2018), from which epidemiological data were extracted. New histological sections were performed for impregnation by the argyrophilic nucleolar organizer regions (AgNOR) technique. The histopathological slides were analyzed by photon microscopy (1,000×), and the nuclei of 100 epithelial cells were photographed to count the number of NORs. Three methods were used for the lesions' histopathological classification [World Health Organization (WHO), Brothwell, and binary system]. The means of NORs were compared with the three histopathological classifications by means of the t or analysis of variance (ANOVA) statistical tests, at a significance level of 5%. According to the WHO classification method, most cases (11-61.1%) had a moderate classification. Evaluations by the Brothwell method showed moderate and mild classification in 50 and 38.9% of cases, respectively. According to the binary system, most cases (10-55.6%) had low risk. The average NORs found in 100 nuclei of each of the 18 lesions ranged from 2 to 4. When crossing the average NORs with the histopathological classification methods of the lesions by means of the t test or ANOVA, no significant relationship was noted. The average of NORs is not associated with the histological classifications of leukoplakias. Thus, the AgNOR method should be used with caution when differentiating different histological grades of leukoplakias. The AgNOR method should be used with caution to determine the clinical treatment of oral leukoplakias, since no agreement was observed between this method and the histopathological classifications available for such lesion.

Does the chromosomal position of 35S rDNA sites influence their transcription? A survey on Nothoscordum species (Amaryllidaceae).

35S ribosomal DNA (rDNA) sites are the regions where the ribosomal genes 18S, 5.8S and 25S, responsible for the formation of the nucleoli, are found. The fact that rDNA sites have non-random distribution on chromosomes suggests that their positions may influence their transcription. To identify if the preferentially transcribed rDNA sites occupy specific position, six species (nine cytotypes) of the genus Nothoscordum were analyzed using two different techniques to impregnate the nucleolar organizer regions (NORs) with silver nitrate. Both techniques strongly stained NORs, but one of them also stained the proximal region of all chromosomes, suggesting the existence of another group of argentophilic proteins in this region. In species with rDNA sites in acrocentric and metacentric chromosomes, sites located on the short arms of the acrocentric chromosomes were preferentially activated. On the other hand, in species with rDNA sites restricted to the short arms of the acrocentrics, all of them were activated, whereas in those species with sites restricted to the terminal region of metacentric chromosomes, the frequency of active sites was always lower than expected. This indicate that, at least in Nothoscordum, the transcription of an rDNA site is influenced by its chromosomal position, and may explain, at least partially, the strongly non-random distribution of these sites in plant and animal chromosomes.

Nucleolar Organizer Region (NOR) Polymorphism in Relation to Interspecific and Intraspecific Variability in Some Indian Anurans-A Review

Nucleolar Organizer Region (NOR) Polymorphism in Relation to Interspecific and Intraspecific Variability in Some Indian Anurans-A Review

Cytogenetic analysis of Rhinella jimi (Stevaux, 2002) (Anura, Bufonidae) from northeastern Brazil

In this work we analyzed the karyotype of Rhinella jimi (Stevaux, 2002) (Anura, Bufonidae)from Picos (Piaui) in Northeastern Brazil. The chromosomes were examined using classicalcytogenetic approaches (Giemsa, C-banding, and Ag-NOR staining). This species has 2n = 22chromosomes, all metacentric or submetacentric. Heterochromatic segments were visualized atthe centromeric region and the nucleolus organizer regions (NOR) were restricted to terminalregions of the short arms in pair 7. There was no evidence of heteromorphic sex chromosomes.The chromosomal analysis of R. jimi allowed us to identify a karyotype that is similar to manyother species of Rhinella, in which the diploid number remains unchanged and without evidencesof structural rearrangements.

Cytopathological tests for early detection of oral carcinogenesis.

The carcinogenesis in the oral cavity occurs as a multistep process and is often preceded by potentially malignant lesions. The main risk factors for the development of oral cancer are smoking and alcohol intake. The current challenge is to identify patients at greatest risk for the development of oral cancer using noninvasive and effective methods. The aim of this study is to evaluate the microsatellite mutations in the 9p21 locus, the cell proliferative activity, the pattern of epithelial desquamation, and the nucleus/cytoplasm ratio of exfoliated epithelial cells. Cytopathological samples were collected from 131 individuals divided into four groups: control (n = 26), alcohol-smoking (n = 32), leukoplakia (n = 38), and the oral squamous cell carcinoma group (OSCC, n = 35). From the cytological scraping, a slide was silver impregnated for Ag-stained nucleolar organizer region analysis and another slide was stained using the Papanicolaou technique. The remaining cells were used for DNA extraction, followed by PCR amplification and capillary electrophoresis. The cell proliferation velocity rate was higher in the leukoplakia and OSCC groups compared with the control group (P < 0.05). The leukoplakia group showed increased anucleated scales, whereas the nucleated superficial predominated in the control group and the parabasal cells in the OSCC group (P < 0.05). An increased nucleus/cytoplasm ratio was detected only in the OSCC group (P < 0.05). The 9p21 locus mutation frequency was higher in the alcohol-smoking and leukoplakia groups. 9p21 analysis and Ag-stained nucleolar organizer region methods are promising for the screening and monitoring of individuals at higher risk for the development of oral cancer.

Argyrophilic nucleolar organizer regions staining for cytology smears in dogs and cats.

The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n=14) and cats (n=12). Acetone, formalin, ethanol and methanol were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May–Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.

Parental species and hybrid descendants of Bacillus (Insecta Phasmatodea) show different patterns of highly amplified, colocalized ribosomal and telomeric sequences

We investigated by dual-color fluorescence in situ hybridization (FISH) with 28S ribosomal and (TTAGG)n telomeric probes all species of the circum-Mediterranean genus Bacillus encompassing bisexual and parthenogenetic taxa, namely the three parental species (B. grandii, B. atticus, B. rossius) and the two derived hybrids (B. whitei, B. lynceorum). Specimens were collected in Italian mainland, Sardinia and Sicily. In all species the presence of colocalized, highly amplified ribosomal and telomeric sequences was demonstrated by the double labelling of the cytological satellites. These satellites varied in size, number and location both among and within species. In B. grandii and B. atticus a maximum of two FISH-labeled locations were observed, whereas in B. rossius and in the two hybrids up to 11 different positions were recorded. Moreover, our investigations showed a significant occurrence of chromosome breakages and rearrangements. The overall meaning of the ribosomal and telomeric sequence colocalization as well as the Nucleolar Organizer Region mobility and activity are discussed in both the ancestors and their hybrid descendants. It is noteworthy that the same trait has been shown in seven additional phasmid species belonging to distantly related genera. This trait could be a shared ancestral character in phasmids.

AgNOR as an effective diagnostic tool for determining the proliferative nature of different types of odontogenic cysts.

Objective:To determine the proliferative nature of different types of odontogenic cysts using silver-stained nucleolar organizer region (AgNOR) staining.Materials and Methods:Forty histopathological sections of each of odontogenic keratocyst, dentigerous, and radicular growth were taken into study. Two sections were produced using the paraffin block of separate cases, one was stained with hematoxylin and eosin and another segment was stained with silver nitrate method.Results:AgNOR staining shows black dots and other parts of nuclei show yellow-brown dots.Conclusion:AgNOR, which stain for NOR-related proteins, corresponds to cell multiplication. Their amount regarding the number of AgNORs per cyst can be a good marker of the proliferative action of the cell in aggressive cystic lesion having malignant potential.

A Study on Immunohistochemical Expression of Progesterone Receptor and Cell Proliferation by AgNOR in Canine Mammary Tumors (CMT)

Mammary gland tumors are the most commonly diagnosed tumors in domestic dogs. Although immunohistochemical methods provide valuable information such as the location and semi-quantitative data of the interested antigens in particular tumors, conventional methods like histopathological diagnosis remain useful and necessary for identification and classification of tumors. In the present study, we combined histopathology with immunohistochemical staining for progesterone receptor (PR) and special staining argyrophilic nucleolar organizer regions (AgNOR) for cell proliferation in canine mammary gland tumors. Twenty-nine dogs with primary mammary gland neoplasms (11 benign and 18 malignant) were included in this study. All 29 tumors were stained with AgNOR but only 15 cases expressed positive PR signals in the nuclei of neoplastic alveolar and ductal epithelial cells. A weak positive correlation (r = 0.2) was observed between PR and AgNOR index of benign tumors and a very weak negative correlation (r = -0.1833) between PR and AgNOR index of malignant tumors suggesting that higher cellular proliferation is associated with low expression of progesterone receptors.

A novel ZZ/ZW chromosome morphology type in Eigenmannia virescens (Gymnotiformes: Sternopygidae) from upper Paraná River basin

Gymnotiformes fishes present a great variation in chromosome numbers. Sternopygidae species are either sex-homomorphic or sex-heteromorphic. This study reports a novel ZZ/ZW chromosome morphology type in Eigenmannia virescens collected from the upper Parana River basin, which presented karyotype composed of 2n = 38 chromosomes, 6 metacentric (m), 10 submetacentric (sm), 4 subtelocentric (st) and 18 acrocentric (a) for males and 5 m, 11 sm, 4 st and 18 a for females, where the Z is a metacentric chromosome and the W is submetacentric chromosome. A single pair of chromosomes carrying the nucleolar organizing regions (NORs) was detected in the acrocentric chromosome pair No. 15 by silver-staining and confirmed by the 18S rDNA-FISH. Multiple 5S rDNA sites were detected in the karyotype, all of them in pericentromeric regions. The heterochromatin was mainly located in the pericentromeric regions of all chromosomes, and a conspicuous accumulation of heterochromatin colocated in NORs sites. Thus, the variation of Z and W chromosome types, associated with different chromosome sex systems (undifferentiated, XX/XY and ZZ/ZW) and different karyotypic formulae, reinforce that E. virescens should be considered a species complex.