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PubMedDate not supplied

High-resolution image cytometry of rat sperm nuclear shape, size and chromatin status. Experimental validation with the reproductive toxicant vinclozolin.

Recent studies have shown that the complex inter-related processes of sperm chromatin organization and nuclear morphogenesis, both of which are important fertility determinants, may be disrupted by chemicals. A high-resolution image cytometry method has been developed, using the fluorescent dye bisbenzimide, for the measurement of 20 features of the sperm nucleus related to size, form and chromatin status in the rat. For the complete set of features measured and from a total of 150 spermatozoa assessed per sample, the overall coefficient of reproducibility was 5%. Then, an experimental validation of the method was carried out in rats chronically exposed to the antiandrogenic reproductive toxicant vinclozolin and control animals. Univariate statistics revealed significant vinclozolin-induced changes for 19 out of 20 morphometric and chromatin features. Stepwise linear discriminant analysis classified correctly 84.3% of the sperm nuclei with only four features selected. The accuracy and reproducibility of the cytometry assessment of the sperm nuclei together with the results of the experimental validation suggest this method may be a new powerful tool for use in reproductive toxicology.

Androgen Antagonists↗
PubMed2005

Melatonin protects against lead-induced hepatic and renal toxicity in male rats.

The present study was designed to investigate the potential protective effect of melatonin against the hepatic and renal toxicity of lead in male rats. Three groups of animals were used in this study (control, lead acetate-treated (100 mg/kg), and lead acetate plus melatonin (10 mg/kg) for 30 days. Levels of lipid peroxidation (LPO) products, superoxide dismutase (SOD) activity, total glutathione (GSH), histopathological changes in the liver and kidneys were investigated. In addition, nuclear area (NA), nuclear volume (NV) and the ratio of nuclear volume/cellular volume (N/C) were measured in the liver. The results revealed increased LPO and decreased SOD, GSH, NA, NV and N/C in the studied organs of lead-treated rats. Histopathological observations showed severe damage in the liver and kidneys. Melatonin co-treatment to the lead-administered rats attenuated the increase of LPO and restored the activity of SOD and levels of GSH as well as the mean values of NA, NV and N/C. Also, the morphological damage in the liver and kidneys was reduced and the tissues appeared like those of controls. The present study suggests that melatonin may be useful in combating free radical-induced damage due to lead toxicity.

Animals↗
PubMed2006

Accelerated nuclei preparation and methods for analysis of histone modifications in yeast.

The continuing identification of new histone post-translational modifications and ongoing discovery of their roles in nuclear processes has increased the demand for quick, efficient, and precise methods for their analysis. In the budding yeast Saccharomyces cerevisiae, a variety of methods exist for the characterization of histone modifications on a global scale. However, a wide gap in preparation time and histone purity exists between the most widely used extraction methods, which include a simple whole cell extraction (WCE) and an intensive histone extraction. In this work we evaluate various published WCE buffers for their relative effectiveness in the detection of histone modifications by Western blot analysis. We also present a precise, yet time-efficient method for the detection of subtle changes in histone modification levels. Lastly, we present a protocol for the rapid small-scale purification of nuclei that improves the performance of antibodies that do not work efficiently in WCE. These new methods are ideal for the analysis of histone modifications and could be applied to the analysis and improved detection of other nuclear proteins.

Buffers↗
PubMed1975

Mitochondriosis of the hepatocytic cytoplasm in rachitic rat liver after calcitonin-treatment (a cytophotometric and morphometric study).

A calcium-rich rachitogenic vitamin-D deficiency and additional doses of calcitonin cause serious changes in cell structure and metabolism, which can be demonstrated by cytophotometry and morphometry. The experiments were made with 9 male juvenile Wistar rats. 6 animals were kept on a vitamin-D deficient diet for 6 weeks and 3 of these animals got injections with salmon-calcitonin S.C. during the last 14 days every second day. The remaining 3 animals, fed with altromin-R standard diet were kept for control purposes only. The morphometric analysis of the liver parenchymal cells was based on the statements by Weibel and the cytophotometric examinations were made according to the Sandritter technique. As a consequence of the altered metabolic condition a great volume reduction of cells and cell nuclei is found in the animals with vitamin-D deficiency, which were treated with calcitonin. In comparison with the control animals the hepatocytes show hardly any tetraploid nuclei, which points to an inhibited polyploidization. The cytoplasm shows an oncocytic transformation with megamitochondria. As reason for this mitochondriopathy an extreme and abnormal growth as well as a mutation of the mitochondrial genom are under discussion. Furthermore the total endoplasmic reticulum (volume and membrane surface), the peroxisomes and the ribosomes are strongly reduced. Due to the serious cell injury the other organelles are also reduced in number and volume, which points to a chronic hypocalcemia.

Animals↗
PubMed1975

Heteropyknosis of the chromosomes in liver cells of different ploidy: a nuclear image study.

The chromatin densities of Feulgen-stained diploid, tetraploid, octoploid and binucleate cells of smear preparations of the liver of female field voles, Microtus agrestis, were examined by means of image analysis. The ratio of the areas of flattened nuclei of 2n, 4n and 8n ploidy was about 1 : 1.62 :2.60 and that of the relative DNA content 1 : 2 : 4. In flattened polyploid nuclei, the chromosomes are more densely arranged than in diploid. In diploid nuclei, absolutely and percentually smaller areas of dark chromatin particles were found than in polyploid nuclei. After correction of the grey values with a density factor, the frequency distribution curves of all ploidy classes were found to be nearly identical. The results show that the percentage of heteropyknotic chromosome material is the same in diploid, polyploid and binucleate liver cells.

Animals↗
PubMed1976

Nuclear size and chromatin concentration in transitional cell carcinoma of the human urinary bladder.

Feulgen-stained cell nuclei from 25 specimens from transitional cell carcinoma of the human bladder were examined by means of cytophotometric scanning measurements. The mean values of nuclear area, DNA-content and chromatin concentration after scanning of a hundred nuclei per specimen were compared with those from a control group consisting of 10 specimens of normal transitional cell epithelium. Within the same ploidy class the more differentiated carcinoma cells showed nuclear enlargement and decrease of mean chromatin concentration. Undifferentiated tumours could have the same or higher mean chromatin concentration as the control group owing to the many highpolyploid nuclei. Undifferentiated tumours contained numerous giant nuclei, whereas these were rare in well differentiated carcinoma. When allowances were made for variations due to fixation and staining measurement of the nuclear area could serve as means of differentiating between normal and cancer nuclei with a high degree of statistic probability. The question whether nuclear enlargement and decreased chromatin concentration represent physiological changes or are entirely or partly the result of chemical carcinogenesis is discussed.

Carcinoma, Transitional Cell↗
PubMed1976

"Non-condensed" and "condensed" chromain in transitional cell carcinoma of the human urinary bladder.

The area and content of "non-condensed" and "condensed" chromatin in smeared Feulgen-stained malignant urothelial cells were determined by means of scanning-cytophotometry. The results were compared with those from similar measurements of benign human transitional epithelial cells. There was no difference between the relative area and content of "non-condensed" and "condensed" chromatin in cancer nuclei and normal urothelial nuclei as far as nuclei of the same size and ploidy class were considered. Within the same ploidy class the relative area and content of "non-condensed" chromatin increased with increasing nuclear size. As increased nuclear size within the same ploidy class is typical for most cancer cells, cancer specimens therefore contained relatively more "non-condensed" chromatin than normal urothelium. Analogously the relative values of "condensed" chromatin decreased in cancer specimens. Only in high-polyploid cancer cells, which occurred more frequently in undifferentiated tumours, a slight decrease of the relative area and content of "non-condensed" chromatin was observed as compared with well differentiated diploid tumour cells. It was in polyploid tumours that the absolute area and content of "condensed" chromatin was increased as compared with diploid normal urothelium. This means that the changes in "non-condensed" and "condensed" chromatin were primarily dependent on nuclear size and total chromatin content and were not found to be a characteristic of cancer nuclei as compared with control nuclei of the same size and ploidy. These findings differ from the results of biochemical analyses of heterochromatin both in cells during carcinogenesis and also in cancer cells, but are in agreement with qualitative and quantitative morphological studies of smeared cancer nuclei.

Carcinoma, Transitional Cell↗
PubMed1987

An in vivo and in vitro study of the embryonic and adult lop mutant congenital cataractous lens.

A detailed histological study of the embryonic and adult congenital cataract Lop mouse lens has been made. A comparison with the congenital cataract (CatFr) mouse lens has shown that the full range of lens anomalies noted in the congenital cataractous mice are very similar. However, the Lop mutant demonstrates these defects to a greater degree in both the embryonic and adult stages. The lens epithelial cells of the Lop adult lens have been cultured to ascertain their in vitro phenotype. These studies have shown that the proliferation pattern and population doubling level are similar to that of the CatFr lens epithelial cells. A comparison with non-cataractous mouse lens epithelial cells demonstrates that the congenital cataractous mice lens epithelial cells have a very limited life span in vitro. An increase in the nuclear diameter with population doubling level was observed in the cultured mouse lens epithelial cells.

Animals↗
PubMed1975

Comparative karyometric studies on small pseudolobuli and hepatomas in thioacetamide induced liver cirrhosis.

In the tumorous phase of TAA-induced cirrhosis of the liver comparative studies on the mitotic index, on the frequency of binucleated liver cells and on nuclear volume showed approximately similar values in the small pseudolobuli and in several hepatomas. Obviously "minimum deviation hepatomas" had developed. Decrease of ploidy characteristic for hepatomas was observed. In thioacetamide hepatomas the phenomenon of quadrupling of the cell nuclei due to dysfunctional nuclear edema occurred whereas the normal nuclei of liver cells can only double in volume in all phases of thioacetamide-intoxication. Particular karyometric aspects of the nuclear-nucleolar metabolism of hepatoma cells following thioacetamide intoxication are discussed.

Animals↗
PubMed1989

Quantitative assessment of the stage of neuronal maturation in the developing human fetal gut--a new dimension in the pathogenesis of developmental anomalies of the myenteric plexus.

There are two main theories of the normal development of the enteric nervous system: the classical theory suggests that the enteric neuroblasts migrate along the alimentary tract in a single cranio-caudal direction. However, the second theory postulates a dual gradient of neuronal development. The present study aims to support the second theory and contribute to the understanding of the pathogenesis of Hirschsprung's disease and its allied gut motility disorders. Using a histomorphometric method, quantitative estimation of the myenteric intraganglionic neuronal development was made in tissues from various selected levels of the gut in 32 normal human fetuses of 11 to 24 weeks ovulation age. A parallel study with special chromatin staining was carried out from these materials to investigate different neurons in the myenteric plexus. Development of intraganglionic neurons was shown to be more advanced in the esophagus, less so in the rectosigmoid and least so in the ileocecal region: the mean values of neuronal and nuclear volume were found in the esophagus greater than in rectosigmoid and ileocecal region as ovulation age progressed. Intraganglionic mitotic figures were detected at all the selected levels of the gut. A decrease of the mitosis index as age progressed was observed. We showed that myenteric neurons followed a dual gradient of development proceeding from both ends to the middle of the gut in mid-trimester human fetuses. Our findings are compatible with the suggestion that any alterations in the fetal gut microenvironment may affect seriously the normal development of a multipotential precursor cell population resulting in various congenital anomalies of the myenteric plexus.

Cell Differentiation↗
PubMed1985

Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells.

The radiosensitive cell populations, such as resting lymphocytes from thymus, spleen, lymph node and blood, have much smaller nuclei (Vn (nuclear volume) approximately 20 to 70 microns3) compared to radioresistant G0 cells from non-lymphoid tissues (liver, kidney, brain, heart; Vn approximately 75 to 2700 microns3). It is suggested that radiation-induced disorganization of nuclear structures and cell pycnosis (interphase death) are promoted in G0 lymphocytes because in normal physiological conditions their nuclei assume a higher degree of chromatin condensation. In contrast, dispersion of chromatin into larger nuclear volumes, such as those of most non-lymphoid G0 cells, may hinder or delay radiation-induced cell death.

Animals↗
PubMed1991

Prognosis of transitional cell bladder cancer: a multivariate prognostic score for improved prediction.

Clinical and histological prognostic factors were evaluated by means of Cox's analysis in 265 bladder cancer patients with a mean followup of 10 years. The parameters studied were obtained from the primary biopsies, which included clinical stage, World Health Organization grade, papillary status, morphometrically measured mean nuclear area, standard deviation of nuclear area, mean nuclear area of the 10 largest nuclei, mitotic activity index and volume corrected mitotic index. In univariate survival analysis all of the parameters predicted survival (p less than 0.001). In Cox's analysis the clinical stage was the most important prognosticator (p less than 0.001) followed by papillary status (p less than 0.001), volume corrected mitotic index (p = 0.011) and nuclear area of the 10 largest nuclei (p = 0.091). In stages Ta to T2, grades 1 to 2 tumors the papillary status (p = 0.001), mitotic activity index (p = 0.021) and T category (p = 0.029) showed independent prognostic value. Among the stages Ta to T1 tumors the papillary status included all of the available prognostic information (p = 0.001). In a separate analysis of histological features in all papillary tumors histological grade (p less than 0.001) and mitotic activity index (p = 0.021) were related independently to survival in Cox's analysis. In papillary stages Ta to T2, grades 1 to 2 tumors (mitotic activity index, p = 0.029) and in papillary stages Ta to T1 tumors (volume corrected mitotic index, p = 0.054) mitotic indexes showed independent prognostic value. In grade 2 tumors the papillary status p = 0.004) and mitotic activity index (p = 0.090) had independent prognostic value. The mitotic indexes predicted progression among stages Ta to T1 tumors (p less than 0.001) and within World Health Organization grades significantly. The combination of prognostic parameters into prognostic scores gave a more accurate estimate of survival than the single parameter approach. The results suggest morphometric grading of bladder tumors. However, papillary and nonpapillary tumors require different grade limits.

Aged↗
PubMed1990

Nuclear morphometry as a predictor of response to therapy in Wilms tumor: a preliminary report.

Present therapy in Wilms tumor is based upon prognosis. Current trends suggest less intensive chemotherapy and/or radiation therapy for children with favorable histological disease who are considered at low risk for recurrence and increasing the amount of therapy for those with unfavorable histological tumors who are at high risk for recurrence. Currently, pathological stage and histological appearance are used to predict prognosis. We tested a new approach. Histological sections of surgical specimens were obtained from 9 children treated for unilateral Wilms tumor. All patients had favorable histological disease, and received postoperative chemotherapy and/or radiotherapy. These specimens were evaluated by nuclear morphometry with the Hopkins Morphometry System to determine if nuclear shape descriptors could distinguish children who responded to therapy from those whose tumor recurred. Nuclear roundness, ellipticity, convexity and bending energy accurately separated the groups. Nuclear morphometry may provide useful information that will aid in predicting which children with Wilms tumor will respond to therapy and warrants further investigations.

Cell Nucleus↗
PubMed1990

Nuclear shape analysis for assessment of prognosis in renal cell carcinoma.

Clinically localized renal cell carcinoma is cured by radical nephrectomy in 47% [stage T3a (II)] to 65% [stage T1, T2 (I)] of the patients. Local recurrence and metastatic disease probably result from undetectable microscopic metastases present at operation. Chemotherapy and immunotherapy may improve cure rates if administered adjuvantly. The outcome of individual patients who share surgical stage cannot be predicted reliably by tumor histology, pathological and/or nuclear deoxyribonucleic acid analysis. Two groups of 10 patients with clinically localized renal cell carcinoma were similar by sex distribution (5 men and 5 women), surgical stage (stages T1 in 1, T2 in 6 and T3a in 3 patients) and age (54.3 +/- 15.2 standard deviation versus 55.8 +/- 8.7 years). Group 1 had no recurrences with a minimum followup of 5 years and a mean followup of 10 years. Group 2 died of metastatic renal cell carcinoma after a mean of 5 years. All neoplastic areas of each paraffin-embedded operative specimen were randomly sampled and the nuclear perimeter of 150 cancerous cells was digitized. There were 25 shape descriptors calculated for each nucleus. All shape descriptors for each patient were described by 19 statistical tests. Nuclear perimeter and area as well as mean nuclear roundness factor failed to separate the 2 groups. Range median quartiles of ellipticities by Fourier analysis, coefficients of variation of chain code minimums and relative means of largest 10 convexity values produced greatest separation (Mann-Whitney-Wilcoxon test p less than 0.001, and variance normalized difference 3.21, 3.29 and 2.83, respectively). These descriptors normalized and summed provided near perfect separation (Mann-Whitney-Wilcoxon test p less than 0.001 and variance normalized difference 3.59). We developed a quantitative nuclear morphometric analysis system that permitted the correct assignment of outcome in 19 of 20 patients. Accurate prediction of prognosis in patients with clinically localized renal cell carcinoma by nuclear shape analysis may allow for selection of patients for adjuvant therapy who have clinically undetectable metastatic disease.

Carcinoma, Renal Cell↗