Avaliação da técnica de coloração AgNOR em testículos de ovinos / Evaluation of the AgNOR staining method in ovine testicles

A coloração pela prata das regiões organizadoras de nucléolos (NORs) é caracterizada por marcar proteínas ligadas ao ácido ribonucleico ribossômico, avaliando a proliferação em células normais ou neoplásicas. Objetivou-se estudar, em testículos de ovinos obtidos em matadouro, a validade do uso da técnica de coloração pela prata (AgNOR) na identificação das regiões organizadoras de nucléolo (NORs) em células saudáveis da linhagem espermatogênica. Utilizaram-se 43 pares de testículos de ovinos mestiços entre seis e 10 meses de idade. Testes de Wilcoxon e Spearman foram empregados, com nível de 5%. As médias das NORs nas células das gônadas direita e esquerda foram, respectivamente: espermatogônia (8,77±1,14 e 9,04±0,96), espermatócitos (4,99±2,00 e 6,20±2,07; P<0,05), Leydig (8,05±2,82 e 7,89±2,29) e Sertoli (8,07±1,88 e 7,61±2,16; P<0,05). Houve correlação (P<0,05) entre os lados para o número de NORs: espermatócitos x Leydig (0,49); espermatócitos x Sertoli (0,49) e Leydig x Sertoli (0,96). Conclui-se ser válido o emprego da técnica AgNOR para avaliar o potencial proliferativo das células saudáveis em testículos de ovinos com prática execução e baixo custo.(AU)

The silver staining technique for AgNOR nucleolar organizer regions (NORs) is characterized by marking proteins linked to the ribosomal ribonucleic acid, evaluating cell proliferation. The aim was to study the validity of the AgNOR staining technique in the testicular cell proliferation in crossbred ovine. Forty-three pairs of ovine testicles between 6 and 10 months old were collected. Wilcoxon and Spearman tests were used with a significance level of 5%. The mean NORs count in cells of the right and left gonads were respectively: spermatogonia (8.77±1.14 and 9.04±0.96), spermatocytes (4.99±2.00 and 6.20±2.07, P<0.05), Leydig (8.05±2.82 and 7.89±2.29) and Sertoli cells (8.07±1.88 and 7.61±2.16; P<0.05). There was a correlation between the mean values for the right and left sides for the number of NORs (P<0.05) between Leydig x spermatocytes (0.49); spermatocytes x Sertoli (0.49) and Sertoli x Leydig (0.96). The study demonstrates that the AgNOR staining technique is indicated to evaluate the cell proliferative potential in ovine testis with practical implementation and low cost.(AU)

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora)

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Morfometría del nucleolo en la diferenciación celular del epitelio mamario / Nucleolus morphometry in the cellular differentiation of mammary epithelium

Antecedentes: El proceso biológico de diferenciación celular es la traducción de múltiples procesos nucleares y citoplasmáticos que determinan cambios complejos y fundamentales en la ultraestructura, bioquímica y fsiología celular, los cuales pueden ser cuantifcados mediante técnicas morfométricas. Objetivo: Evidenciar en términos cuantitativos y morfológicos las variaciones experimentadas por los nucleolos pertenecientes a células mamarias de la línea HC11 tanto normales como en mecanismo de diferenciación. Método: Se estudió a nivel de la microscopía electrónica de transmisión los tipos celulares en etapa de proliferación (HC11 GM) en comparación con células en estadio de diferenciación (HC11 IM), cuantifcando las variaciones de los nucleolos y su relación con estructuras involucradas en síntesis proteica. Resultados: Se evidencian diferencias estadísticamente signifcativas referentes al área, volumen y longitud entre los nucleolos pertenecientes al tipo celular normal-proliferante y el que se encuentra en proceso de diferenciación. Conclusión: Las células mamarias en proceso de diferenciación presentan una notable disminución de sus nucleolos, y sus ribonucleoproteínas constitutivas generarán básicamente ribosomas adheridos al retículo endoplasmático rugoso, sintetizando proteínas de exportación.

Background: The biological process of cell differentiation is the traslation of multiple nuclear and cytoplas-mic processes that determine complex and fundamental changes in ultrastructure, biochemistry and cell physiology, which can be quantifed using morphometric techniques. Objective: To show in quantitative and morphological terms changes experienced by the nucleolus belonging to HC11 line mammary cells both, in proliferating and differentiation process. Methods: A study at the level of transmission electron microscopy of cell types in stage of cell proliferation in comparison with stage of differentiation was designed to quantify variations of nucleolus and their relation to structures involved in protein synthesis. Results: Marked differences in the area, volume and length of the nucleolus were found between normal-proliferating cell types and those in mechanism of differentiation. Conclusion: The mammary cells in differentiation process show a dramatic decline in its nucleoli and their ribonucleoproteins generate basically ribosomes attached at endo-plasmic reticulum, synthesizing export proteins.

Cytogenetical and ultrastructural effects of copper on root meristem cells of Allium sativum L

Different copper concentrations, as well as different exposure times, were applied to investigate both cytogenetical and ultrastructural alterations in garlic (Allium sativum L.) meristem cells. Results showed that the mitotic index decreased progressively when either copper concentration or exposure time increased. C-mitosis, anaphase bridges, chromosome stickiness and broken nuclei were observed in the copper treated root tip cells. Some particulates containing the argyrophilic NOR-associated proteins were distributed in the nucleus of the root-tip cells and the amount of this particulate material progressively increased with increasing exposure time. Finally, the nucleolar material was extruded from the nucleus into the cytoplasm. Also, increased dictyosome vesicles in number, formation of cytoplasmic vesicles containing electron dense granules, altered mitochondrial shape, disruption of nuclear membranes, condensation of chromatin material, disintegration of organelles were observed. The mechanisms of detoxification and tolerance of copper are briefly discussed.

p53 and MDM2 protein expression in actinic cheilitis

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Analysis of the nucleolar cycle in the seminiferous epithelium of rodents

The nucleolus is a subcompartment of the nucleus and the site of ribosome biogenesis. During the mitotic and meiotic cell cycles, a disorganization and later reorganization of the nucleolar material occur, an event called nucleologenesis. In the spermatogenesis of mammals and other vertebrates, there is evidence of the disorganization of the nucleolus at the end of meiosis I, which supplies material for the cytoplasmic formation of an organelle called the “chromatoid body” (CB). The CB is a structure characteristic of spermatogenic cells and seems to be responsible for RNA metabolism in these cells and for some events of spermiogenesis, such as the formation of the acrosome, cellular communication between spermatids, and the formation of the spermatozoon middle piece and tail. The aim of this paper was to obtain information about the cytochemical and ultrastructural nature of the nucleolar cycle and the distribution of cytoplasmic RNAs in the seminiferous tubule cells of Rattus novergiucus, Mus musculus and Meriones unguiculatus. The testis was fixed in Bouin and Karnovsky solutions for conventional histological analysis and for cytochemical study that included: periodic acid-Schiff, hematoxylin-eosin, Feulgen reaction, silver-ion impregnation, Gomori’s reticulin stain, toluidine blue, modified method of critical electrolyte concentration, and basic and acid fast green. The blocks of testis fixed in glutaraldehyde were used for ultrastructural analysis by transmission electron microscopy. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All the techniques used provided information on the origin and function of the CB in the spermatogenic cells. Therefore, considering the persistence of the RNA and nucleolar ribonucleoproteins during spermatogenesis of Rattus novergicus, Mus musculus and Meriones unguiculatus, our findings corroborate the statement that these molecular complexes are very important in the spermiogenesis phases...

Subcellular localization of cadmium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry.

The ultrastructural investigation of the root cells of Allium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver