AgNOR Technique Research Articles

Türkiye'ye endemik Squalius cappadocicus Özuluğ and Freyhof, 2011'un sitogenetik özellikleri

In this study, a detailed chromosome analysis of the endemic Cappadocian Chub, Squalius cappadocicus in Melendiz Stream (Aksaray) was performed. The standard Giemsa staining, C-banding (CBG and CB-DAPI), and Ag-NOR technique were applied. The diploid chromosome number was 50; its karyotype formula was 14M + 16Sm + 10St + 10A. Heteromorphic sex chromosomes weren’t detected in the karyotype of the studied specimens. The number of all chromosomal arms (NF) was 90. In the standard C-banded and CB-DAPI karyotype of the species, dark C-bands were observed in the centromeric region of some bi-armed and acrocentric chromosomes, while slightly centromeric or pericentromeric C-bands were detected in some chromosomes. Three different active Ag-NORs, which were hemizygous, were detected in all samples examined. Two of these active NORs were detected in the bi-armed and the other in the acrocentric chromosome short arm. The Ag-NOR number of this species was evaluated as a feature that distinguishes it from other Squalius species in Türkiye.

Karyotypic variation of two populations of the small freshwater stingray Potamotrygon wallacei Carvalho, Rosa & Araújo 2016: A classical and molecular approach.

Potamotrygoninae comprises a group of Neotropical fishes with an ancient relationship with marine environments. In the last few years, 11 new Potamotrygon species were described, including Potamotrygon wallacei Carvalho, Araújo e Rosa 2016. Cytogenetic data about this species are limited to classical markers (Giemsa, C-Banding and Ag-NOR techniques), these studies highlighted a rare sexual chromosome system XX/X0 with males presenting 67 chromosomes and females 68 chromosomes. The classical analyses performed here reveled populational variation in the karyotype formula, as well as, in the heterochromatin regions. Besides the classical markers, our molecular experiments showed multiple sites for 18S rDNA sequence (including in the X chromosomes) and single sites for 5S rDNA sequence, we did not find interstitial telomeric sequences. In addition, (AC)15, (AG)15, and (CAC)15 microsatellites showed association with the several autosome pair, and the (GT)15 clutters were found in only one population. On the other hand, (GATA)4 sequence showed association with the sexual chromosomes X in all males and females analyzed. Our results showed that pericentric inversions, in addition to fusions, shaped the karyotype of P. wallacei once we found two populations with distinct karyotype formula and this could be a result of the past events recovered by our modeling experiments. Besides, here we described the association of 18S and (GATA)4 motifs with sexual chromosomes, which indicated that these sequences had a novel in the differentiation of sexual chromosomes in P. wallacei.

Karyotypes and ZZ/ZW Sex Chromosome System of Endemic Squalius recurvirostris (Leuciscinae, Cyprinidae) in Turkey

In this study, detailed cytogenetic properties of the endemic Akşehir chub, Squalius recurvirostris, distributed in the basin of the Ilgın (Çavuşçu) Lake basin was investigated by applying the standard Giemsa staining, C-banding and Ag-NOR technique. Diploid chromosome number is (2n) 50. A pair of heteromorphic chromosomes was detected in the karyotype in females. However, this difference was not observed in males. Therefore, it was determined that the heteromorphic chromosome pair in females were ZZ/ZW sex chromosomes. The Z chromosome in females is large acrocentric, and the W chromosome is medium-sized submeta/subtelocentric. The number of all chromosomal arms (NF) is 90, and the fundamental number of autosomal arms (NFa) is 88 in the karyotyped of the individuals studied. Some bi-armed and acrocentric chromosomes have centromeric C-bands in the chromosome set. The slightly centromeric or pericentromeric C-bands are observed in some chromosomes, while other chromosome pairs are C-negative. In the individuals studied two active NORs were detected. Both of these NORs were hemizygous and associated with a large C-heterochromatin region. One of the NORs was detected on the entire long arm of the submetacentric chromosome pair and the other on the entire short arm of another submetacentric chromosome pair.

The value of PAP and AgNOR techniques in identification of bacterial infections and Cytomorphological changes in sputum of Sudanese pango Smokers

The value of PAP and AgNOR techniques in identification of bacterial infections and Cytomorphological changes in sputum of Sudanese pango Smokers

Semantic Segmentation for the Detection of Very Small Objects on Cervical Cell Samples Stained with the AgNOR Technique

Semantic Segmentation for the Detection of Very Small Objects on Cervical Cell Samples Stained with the AgNOR Technique

The value of PAP and AgNOR techniques in identification of bacterial infections and Cytomorphological changes in buccal cavity of Sudanese Hookah Users

The value of PAP and AgNOR techniques in identification of bacterial infections and Cytomorphological changes in buccal cavity of Sudanese Hookah Users

Study of Association of AgNOR vs Ki-67 in Thyroid Lesions

The thyroid is an important endocrine gland, which plays a major role in the functions of almost all normal metabolic processes. In present study, by focusing the sections at very high magnification (100x, oil immersion level) AgNOR dots are visible as black dots inside the nucleus. Our studies which have reported that thyroid lesions with less AgNOR counts are more likely to regress as compared to those with high AgNOR counts and that showed its prognostic significance. Thus, the AgNOR technique can be useful as a supportive tool in both the prognosis and therapeutic value in thyroid lesions most commonly in malignant lesions that are stained by routinely performed haematoxylin and eosin. The Present Study is aim to find out the prevalence of thyroid lesions in the patients and also to Study and find out the different types of neoplastic and non- neoplastic thyroid lesions and their relation with AgNOR and Ki-67 proliferation makers. The Present Study focuses on to evaluate the association of AgNOR vs Ki-67 in the prognostic value of thyroid lesions.

Molecular cytogenetics insights in two pelagic big-game fishes in the Atlantic, the tarpon, Megalops atlanticus (Elopiformes: Megalopidae), and the sailfish, Istiophorus platypterus (Istiophoriformes: Istiophoridae)

Abstract Some pelagic and usually large sized fishes are preferential targets for sport and commercial fishing. Despite their economic importance, cytogenetic data on their evolutionary processes and management are very deficient, especially due to logistical difficulties. Here, information for two of such charismatic species, the tarpon, Megalops atlanticus (Elopiformes: Megalopidae), and the sailfish, Istiophorus platypterus (Istiophoriformes: Istiophoridae), both with a wide Atlantic distribution, were provided. Cytogenetic data were obtained using conventional methods (Giemsa staining, Ag-NORs technique, and C-banding), base-specific fluorochrome staining and fluorescence in situ hybridization (FISH) with rDNA probes. Megalops atlanticus has 2n = 50 chromosomes, all acrocentric ones (NF = 50), while Istiophorus platypterus has 2n = 48 chromosomes, 2m + 2st + 44a (NF = 52). Megalops atlanticus populations from the South Atlantic and Caribbean share identical karyotypic patterns, likely associated with gene flow between them. In turn, I. platypterus presents karyotype similarities with phylogenetically close groups, such as Carangidae. The chromosomal characteristics of these species highlight their independent evolutionary paths. Additionally, the current data contribute to knowledge of new aspects of pelagic fish fauna and will support further comparative studies with congeneric species, clarifying evolutionary karyotype trends of these fish groups.

Overview on Karyotype Stasis in Atlantic Grunts (Eupercaria, Haemulidae) and the Evolutionary Extensions for Other Marine Fish Groups

Karyotype stasis, characterized by the absence of changes in chromosome number, genomic structure, and ploidy, is a recurrent condition in many biological groups, such as plants, amphibians, birds, and fishes. In fishes, the Percomorpha clade (>17,000 species), especially, its largest series, Eupercaria (>6,000 species and 161 families), shows an extensively shared karyotype with 2n=48 acrocentrics; the phylogenetic extension in this series is still unknown. Haemulidae (grunts) .a bounds the zoogeographic marine provinces of the Caribbean and Brazil, has a variable effect on the gene flow in diverse fish groups. Geographically, this barrier is located inside the distribution area of several fish species of Atlantic, allowing the examination of its role on the eventual karyotype differentiations in the populations of grunts. In this sense, cytogenomic analyses were carried out in eight haemulid species by conventional cytogenetic approaches (Giemsa staining, C-banding, and Ag-NORs technique), staining with base-specific fluorochromes, and two-color fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. Additionally, we performed an exhaustive survey on the cytogenetic data of species from the Eupercaria series to assess the general context of karyotype divergences. Haemulid species and populations showed a pronounced sharing of karyotypes with 2n=48a, simple NORs, and reduced centromeric heterochromatin, apart from recurrent patterns of ribosomal 5S and 18S rDNA sites. Karyotype similarities persisted at intra- and congeneric levels in the Haemulidae family, indicating accentuated syntenic conservatism through large divergence periods. Stable karyotype patterns were extensively present in the majority of members from Eupercaria (88% of the clades in this series). These results indicate that karyotype stasis, which is found in a large spectrum of marine fishes, is a multifactorial process in terms of phylogenetic, biological, and biogeographic contexts.

Relationship between Canine Lymphocyte AgNOR Counts and Haematological Indices of Health

Abstract A modified agyrophil technique was applied to peripheral blood smears to determine the mean AgNOR counts (MAC) of lymphocytes and ultimately assess the state of the lymphoid system in various clinical conditions of dogs. Fifty dogs, from clinically normal to pets with leukaemia, presented to the Veterinary Teaching Hospital, were recruited. Blood smears from each dog were stained with routine Romanowsky and modified agyrophil stains. Signalment, clinical diagnoses and hematologic parameters of the dogs were related to the MAC. An AgNOR proliferative index (AgPI) — percentage of lymphocytes with 3 or more AgNORs, was determined, and correlated with MAC. The statistical significance was determined at P < 0.05. MAC ranged from 1.17 in clinically healthy patients to 6.00 in leukaemic patients. The MAC was 2.00 in patients (n = 26) with lymphocyte counts within reference intervals (900—2400 per microliter); 2.23 in patients (n = 4) with lymphopenia; 2.18 in patients with lymphocytosis (n = 18) and 4.73 in patients (n = 4) with lymphocytic leukemia. Also, the MAC was 2.00 in non-anemic dogs while it was 2.47, 2.49 and 3.06 in patients with mild, moderate and severe anaemia, respectively. The MAC correlated strongly with AgPI (r = 0.91). The ancillary AgNOR technique provides a cheaper, more rapid and sensitive tool than routine lymphocyte counts in assessing the state of lymphoid proliferation in a variety of conditions in the dog.

Vitrification of ovarian tissue of Brazilian North-eastern donkeys (Equus asinus) using different cryoprotectants.

The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3M or 6M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3M+EG 3M). Ten pairs of ovaries from Brazilian north-eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid-surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p<0.05). When comparing treatments, the use of DMSO 3M (81.7±37.5%), EG 3M (83.7±27.4%) and the combination of both DMSO 3M+EG 3M (81.8±46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6M (69.8±16.5%) and EG 6M (72.3±18.0%; p<0.05). When vitrified using the DMSO+EG combination, a higher percentage (62.5±29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p<0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3M+EG 3M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3M+EG 3M (p>0.05). We concluded that the combination DMSO 3M+EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.

Nucleolar activity in Triticum x Thinopyrum hybrids with different ploidy level

The activity of nucleolar organizer regions (NORs) and the presence of ribosomal DNA (rDNA) zones were studied in two Triticum x Thinopyrum hybrids: a hexaploid hybrid with 2n = 42 chromosomes, named trigopiro SH16 INTA, and a decaploid hybrid with 2n = 56 chromosomes, named trigopiro Don Noé INTA. The use of the pTa71 probe revealed the presence of 10 rDNA signals in both hybrids, whereas the Ag-NORs technique showed 10 signals in SH16 and 8 in Don Noé. We concluded that all trigopiro SH16 INTA NORs are active and that the activity of one NOR pair of trigopiro Don Noé INTA is suppressed. Therefore, the amphiplasty phenomenon is present in trigopiro Don Noé INTA but not in trigopiro SH16 INTA.

How Useful is the AgNOR Staining Method for the Diagnosis of Salivary Gland Tumors?

Objectives: The purpose of this study was to determine the numbers and mean areas of the AgNOR dots in the 3 most common salivary gland tumors. Materials and Methods: One hundred and seventy paraffin blocks histologically diagnosed as pleomorphic adenoma, adenoid cystic carcinoma, and mucoepidermoid carcinoma were selected from the archives of the pathology department of Loghman hospital, Tehran, Iran and then cut and stained using the standard AgNOR method. The number and area of AgNOR dots were evaluated. Chi-square and Pearson correlation (2-tailed) tests were used for statistical analysis. The significance level was P < 0.05. Results: The chi-square test showed significant differences between tumor types and the numbers of AgNORs (P = 0.000) and between tumor types and areas of AgNORs (P = 0.000). Conclusions: The AgNOR technique can be used to diagnose salivary gland tumors.

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.

New insights into karyotypic relationships among populations of Astyanax bockmanni (Teleostei, Characiformes) of different watersheds.

The fish constitute about 50% of all vertebrates, including a wide morphological and biological diversity, where the genus Astyanax is the most common and diverse, as described in virtually all freshwater environments. By occupying a basal position in the phylogeny of vertebrates, fish are an extremely favorable group for cytogenetic and evolutionary studies. The karyotype found in genus Astyanax diversity may involve a number of polymorphisms, which may be related to ploidy and karyotypic macrostructure, presence of B chromosomes, heterochromatin polymorphisms, and location of ribosomal genes. Nevertheless, the relationship between populations of this species is still poorly studied. Thus, the present work aimed to investigate karyotype variation, chromosomal relationships, and the behavior of 5S and 18S ribosomal genes in six populations of Astyanax bockmanni. The results confirmed the diploid number of 50 chromosomes in all the populations sampled, with the occurrence of one supernumerary chromosome in just one of them. In addition, all populations showed divergent patterns of constitutive heterochromatin and repetitive nucleolar sites. The fluorescence in situ hybridization (FISH) technique using 5S and 18S rDNA probes revealed distinct patterns of distribution for these conserved genes, while 5S rDNA genes were found located in two chromosome pairs, the 18S genes showed multiple marks dispersed in the genome characterizing an inter and intraindividual polymorphic behavior, as previously reported to occur with the utilization of the Ag-NOR technique. Thus, besides minor modifications observed in chromosome morphology, the populations of A. bockmanni analyzed revealed a preserved macrostructural feature, especially concerning to the diploid number; on the other hand, differences in microstructural characteristics indicated by the nucleolus organizer region (NOR) location, constitutive heterochromatin patterns, and distribution of ribosomal genes along the genome were clearly evident in the populations from different river basins, even located at short distances.

Genotoxicity evaluation of environmental pollutants using analysis of nucleolar alterations.

Nucleolar alterations resulting from the action of either chemical or physical agents can serve as important genotoxicity biomarkers. In this study, the efficiency of AgNOR banding technique to identify the presence of nucleoli in micronucleus and assess nucleolar alterations in aberrant cells of Allium cepa was evaluated. Seeds of this plant were exposed to both water samples from a river that receives untreated urban effluent and to the trifluralin herbicide (0.84mg/L concentration), both analyzed in two different seasons (summer and winter seasons). Samples induced significant frequencies of chromosomal and nuclear aberrations and micronuclei, as observed in cells submitted to conventional chromosomal staining. The herbicide caused a significant increase in the number of nucleoli and micronuclei, interpreted as due to the elimination of excessive nucleolar material resulting from polyploidization. The use of the AgNOR technique enabled the identification of both the presence of the nucleolus in some micronuclei and the nucleolar organizer region (NOR) behavior of aberrant cells. The NOR-banding technique showed to be an efficient tool for studying the genotoxic effects caused by a xenobiotics and a complex environmental sample.

Argyrophilic nucleolar organizing region associated protein synthesis for cytologic discrimination of follicular thyroid lesions

Fine needle aspiration biopsy (FNAB) of the thyroid gland is an important tool for preoperative diagnosis; however, its benefit is limited for follicular lesions. Nucleolar organizer regions (NORs) are ribosomal gene regions that stain with silver (Ag) when they are active. These regions can be used to differentiate neoplastic and non-neoplastic lesions. We used a new AgNOR technique to investigate FNAB of cases diagnosed as follicular adenoma and carcinoma. Fourteen cases of follicular thyroid carcinoma (FTC) and 28 cases of thyroid follicular adenomas (FA) were stained using the silver NOR-associated protein (AgNOR) technique. One hundred nuclei per sample were examined, AgNORs were counted, and the total AgNOR area/nuclear area (TNORa/Na) ratio of each cell was calculated. We found that cases with FTC had significantly higher TNORa/Na than cases of FA. Also, cases with FTC had significantly higher AgNOR counts than cases with FA. AgNOR counting may help discriminate FTC and FA by routine cytopathology before surgery.

Analysis of silver stained nucleolar organizing regions in odontogenic cysts and tumors.

Objective:The present study aimed to investigate the probable differences in cell proliferation index of odontogenic cysts and tumors by means of a comparative silver stained nucleolar organizing region (AgNOR) quantification.Study Design:This descriptive cross-sectional study was done on archival paraffin blocks (n = 62), consisting of 10 odontogenic keratocysts, 10 dentigerous cysts, 10 radicular cysts, 10 conventional ameloblastomas, 10 adenomatoid odontogenic tumors, 10 calcifying epithelial odontogenic tumors and 2 ameloblasic carcinomas.Results:The mean AgNOR count of odontogenic cysts was 1.709 and the benign odontogenic tumors was 1.862. Highest AgNOR count was recorded in odontogenic keratocyst and lowest was seen in radicular cyst. Statistically significant difference in AgNOR counts of ameloblastoma and adenomatoid odontogenic tumor, amelobalastoma and calcifying epithelial odontogenic tumor, benign odontogenic tumors and ameloblastic carcinoma were seen. AgNORs in ameloblastic carcinoma were more in number and more widely spread.Conclusion:AgNOR technique may be considered a good indicator of cell proliferation in odontogenic cysts and tumors.

Polymorphism of Cytogenetic Markers in Wild and Farm Red Fox (Vulpes vulpes) Populations

Analysis of the origin of domestic animals is of wide interest and has many practical applications in areas such as agriculture and evolutionary biology. Identification of an ancestor and comparison with the domesticated form allows for an analysis of genetic, physiological, morphological and behavioral effects of domestication. Because fox breeding has been an ongoing process for over a century, differences are expected between farm and wild populations at the chromosomal level. The aim of this work was to analyse polymorphisms at the chromosomal level in foxes raised on farms and those living in the wild. Blood samples and lung tissue served as the experimental material and were obtained after slaughter of 35 foxes, including 28 breeding animals and 7 wild animals. The classical cytogenetic method was used including AgNOR technique, as well as molecular methods such as fluorescence in situ hybridization (FISH), and primed in situ labeling (PRINS). Analysis of the number of B chromosomes showed the presence of polymorphisms in foxes from both studied populations, but there was no correlation between the number of B chromosomes and the origin and gender of particular animals. An analysis ofactive nucleolar organizers showed the presence of a large number of polymorphisms and a tendency towards reduction of the number of NORs in the captive-raised population.

Cellular proliferation markers in peripheral and central fibromas: a comparative study

Objective:To perform a comparative study of the cellular proliferation in the peripheral and central fibromas. Material and Methods:Immunohistochemistry for PCNA and the AgNOR technique were performed in 9 cases of peripheral odontogenic fibroma (POF), in 4 cases of odontogenic fibroma (OdF), in 8 cases of peripheral ossifying fibroma (PEOF) and 7 cases of ossifying fibroma (OsF). The Kruskal-Wallis and Mann-Whitney tests were used for the statistical analyses. Results:Mesenchymal component of the central lesions presented a higher mean number of AgNOR per nucleus and PCNA index than did the peripheral lesions (P≤0.05). The mean number of AgNOR per nucleus in the epithelial component proved to be higher in the OdF than in the POF (P≤0.05). The mesenchymal and epithelial components presented similar mean numbers of AgNOR per nucleus and PCNA index in the OdF, as well as a similar mean number of AgNOR per nucleus in the POF.Conclusions:The mesenchymal component may well play a role in the differences between the biological behaviour of the central lesions as compared to the peripheral lesions. Moreover, considering that the epithelial and mesenchymal components in odontogenic fibromas presented a similar proliferation index, more research is warranted to understand the true role of the epithelial components, which are believed to be inactive in nature, as well as in the development and biological behaviour of these lesions.