Microspectrophotometry of cell nuclei stained with the Feulgen reaction. IV. Formation of tetraploid nuclei in rat liver cells during postnatal growth

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

MICROSPECTROPHOTOMETRY OF CELL NUCLEI STAINED WITH THE FEULGEN REACTION : IV. FORMATION OF TETRAPLOID NUCLEI IN RAT LIVER CELLS DURING POSTNATAL GROWTH

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

The variation with age of nuclear phosphoproteins released during micrococcal-nuclease digestion and nucleosomal phosphoproteins in three cell types from rat liver.

The nucleosomal non-histone phosphoproteins, and phosphoproteins released during the digestion of nuclei by micrococcal nuclease, were studied in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2- and 4-month-old rats with increasing proportions of parenchymal tetraploid nuclei. Qualitative and quantitative differences in nucleosomal phosphoprotein band patterns were found among different types of nuclei and ages. More phosphoprotein bands were found in nucleosomes derived from parenchymal than stromal nuclei. The number of phosphoproteins released during micrococcal-nuclease digestion increased with age for parenchymal nuclei. The significance of these results, considered in conjunction with the increase of DNA repeat length and decrease of nuclease accessibility with age, is discussed.     

The variation with age of the structure of chromatin in three cell types from rat liver.

The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, was studied by digestion with micrococcal nuclease and pancreatic deoxyribonuclease in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2-and 4-month-old rats with a high proportion of tetraploid nuclei. Digestion by micrococcal nuclease allowed the estimation of DNA-repeat length in chromatin. Parenchymal nuclei have shorter repeat length than stromal nuclei and DNA-repeat length increases with the age in all three nuclei populations. The kinetics of digestion by micrococcal nuclease showed that nuclei with shorter repeat length are more sensitive to micrococcal nuclease and that the sensitivity of chromatin decreases with age for all the types of nuclei in this study. The kinetics of digestion by pancreatic deoxyribonuclease showed that sensitivity of chromatin is related to the repeat length and that the sensitivity decreases with the ages.     

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity.The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes.By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [³H]thymidine injection it was found that the label moved largely into the tetraploid compartment.Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation.The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Endopolyploidy in Diploid and Tetraploid Maize (Zea mays L.)

Nuclei from different tissues such as stem, mesocotyl, nodalroot and root tip of diploid and tetraploid maize were isolated,stained with propidium iodide and passed through an EPICS-751flow-cytometer cell sorter. Variations in flow histograms wereobserved in different tissues. Stem tissues of both the diploidand tetraploid had two peaks representing G1 and G2 somaticnuclei. The remaining tissues in both the diploids and tetraploidsexhibited three peaks. The first peak observed in these tissuesrepresents G1 somatic nuclei of the lowest ploidy level. Thesecond peak represent G2 somatic nuclei of the lowest ploidylevel+G1 somatic nuclei of the next ploidy level. The thirdpeak represents G2 of the higher ploidy level+G1 somatic nucleiof the next higher ploidy level. Statistically significant differenceswere observed between the diploid and tetraploid maize tissueswith respect to nuclei distribution in the higher ploidy levelpeaks implying variation in the degree of endopolyploidy inthe diploid and tetraploid maize. The results of this studysuggest that the amount of endopolyploid observed in maize genotypeshas an effect on their overall agronomic performance under thefield conditions.Copyright 1993, 1999 Academic Press Zea mays L., maize, endopolyploidy, diploid, tetraploid, flow cytometry     

Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)?

Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei.     

Flow cytometric analysis of mouse hepatocyte ploidy

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

Biochemical,morphological and flow cytometric evaluation of the effects of hexachlorobenzene on rat liver

This study investigated the ability of HCB (0.1% in the diet for 15 days) to cause early changes in the cellular ploidy of rat liver. Treatment caused marked hepatomegaly, increase of microsomal proteins and cytochrome P-450 content and reduction of hepatocyte microviscosity. Microscopic examination showed that the hepatocytes were enlarged, with hyaline cytoplasm and vacuoles. The size distribution of the isolated hepatocytes showed a larger percentage of bigger cells. Flow-cytometric DNA/protein analysis was performed on whole (fixed) cells and on nuclei. From the combined results of both analyses it was possible to exclude significant changes in the percentages of diploid, mononucleated tetraploid, binucleated tetraploid and octoploid hepatocytes. The DNA and protein content of each subpopulation remained unchanged. Our results suggest that HCB does not cause early diploidization of liver cells and that hepatomegaly and cytochrome P-450 induction seem not to be correlated with effects on total DNA and total protein contents.Abbreviations HCB hexachlorobenzene - PI propidium iodide - FITC fluorescein isothiocyanate - DN diploid nuclei - SN 2N-4N nuclei in S-phase - TN tetraploid nuclei - DC diploid cells - SDC 2N-4N diploid cells in S-phase - TC tetraploid cells - STC 4N-8N tetraploid cells in S-phase - OC octoploid cells - MDC mononucleated diploid cells - SMDC mononucleated diploid cells in S-phase - BOC binucleated octoploid cells - SBTC binucleated tetraploid cells in S-phase - BTC binucleated tetraploid cells - MTC mononucleated tetraploid cells.     

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.     

Image cytometric measurements of diploid,triploid and tetraploid fish erythrocytes in blood smears reflect the true dimensions of live cells

Comparative image cytometry of erythrocytes of diploid and triploid tench Tinca tinca L. and evolutionary tetraploid sterlet Acipenser ruthenus L. was performed on whole live unstained cells, live cells with stained nuclei and on stained fixed whole cells and their nuclei to test if erythrocyte measurements made from blood smears reflect the true dimensions of live cells. Nuclear area and perimeter were the best ploidy level predictors distinguishing accurately among live and fixed diploid, triploid and tetraploid cells, without significant differences between live and fixed cells within a ploidy level. Redundancy analysis revealed insignificant marginal effect of fixation (explained 2.3% of variation, F=0.804), whereas the effect of ploidy level was highly significant (explained 50.6% of variation, F=34.874). The erythrocyte measurements of diploid, triploid and tetraploid fish erythrocytes and their nuclei made from blood smears reflect the true dimensions of live cells, and the fixation procedure did not substantially affect their predictive value for ploidy level determination.     

Quantitative cytophotometric studies on polyploid liver cell nuclei of frog and rat

Summary Nuclei were isolated from fixed liver tissue of diploid and triploid Rana pipiens and of rat. While the frog liver nuclei present a single ploidy class on the basis of Feulgen absorption measurements, rat liver contained diploid, tetraploid, and some octoploid nuclei. Nuclear areas within single ploidy classes varied over wide ranges, especially in the frog material. The mode of this variation was dependent on ploidy. Microspectrophotometric measurements of several protein components were compared to ploidy and nuclear volume. General protein methods indicated a linear relationship to nuclear volume. Protein-bound sulfhydryl and disulfide groups were not related to nuclear volume, but could be related to ploidy. Protein tyrosine values showed a partial dependence on both factors.This investigation was supported by a grant from the U.S. Public Health Service, GM-10003-03.Post-Doctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, 5T1 GM 1142-03.Supported by a Career Development award from the National Institute of Child Health and Human Development, K3-DH-6176-03.     

Occurrence of ploidy shift in a strain of the imperfect yeast Candida albicans

A clinical isolate of Candida albicans, a member of the Fungi Imperfecti, was polyploid as shown by the fact that it contained two kinds of nuclei, one of diploid and one of tetraploid DNA content. These determinations were made by fluorescence microscopy-photometry. The nucleus-associated organelles (NAOs), or spindle pole bodies, of yeast cells in this isolate were classified into two groups, one diploid and the other tetraploid, according to their dimensions as determined by serial thin-sectioning electron microscopy. A ploidy shift from diploid to tetraploid was found in individual cells of a culture of this isolate undergoing diphasic growth in minimal salts medium. A process of shift-down or reduction of ploidy from tetraploid to diploid was also observed by electron microscopy during these growth conditions: this appeared to occur in large cells which showed multiple spindle formation during nuclear division, a phenomenon apparently similar to the process of meiosis II during sporogenesis of Saccharomyces cerevisiae, but differing in that it produces diploid daughter nuclei by the vegetative process.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

Flow cytometric analysis of isolated rat liver nuclei during growth

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.     

Ploidy levels and the number of nuclei in cardiomyocytes of the lamprey and fish

It is well known that polyploidization of cardiomyocytes (CMC) is an essential component of heart growth in the warm-blooded vertebrates. Using the Feulgen cytophotometry of alkali-dissociated cells, we determined the ploidy in CMC of the lower vertebrates: lamprey Lampetra fluviatilis (Cyclostomata), skate Bathyraja maculata (Chondrostei), sterlet Acipenser ruthenus, and Russian sturgeon Acipenser güldenst?dti (Ganoids), as well as paradise fish Macropodus opercularis, Amur sleeper Perccottus glehni, and Atlantic salmon Salmo solar (Teleostei). The data obtained have demonstrated a wide variety in CMC ploidy of both cyclostomata and fishes. About 85% of the lamprey CMC contain 2 or more (up to 17) nuclei per cell; with 90 and 10% of the nuclei being, respectively, diploid and tetraploid. Hearts of the skate and sturgeons contain mononucleated diploid CMC. In the perch-like fishes, mononucleated diploid and mononucleated tetraploid CMC make, respectively, 95 and 5%. The salmon heart contains near 50% of mononucleated diploid CMC, 13% of mononucleated tetra- and octaploid CMC, the rest CMC being multinucleated (up to 6 nuclei per cell). In all the examined species, the increased nuclear ploidy is accompanied with a significant increase in the nuclear volume. The number of nucleoli per nucleus does not correlate with the nuclear ploidy level. Evolutionary aspects of CMC polyploidy in chordates are discussed.     

The nuclear dry weight of liver cells from patients with virus hepatitis and from controls.

Interferometric investigations were performed at liver cell nuclei isolated in 70 per cent glycerol. In 11 patients with virus hepatitis and seven patients without liver disease the nuclear dry weight of liver cells obtained by liver biopsy was determined by interferometry. The average nuclear dry weight of diploid liver cells from controls was 39.9 pg while an average value of 45.4 pg was found for patients with hepatitis. The corresponding nuclear volumes were 241 and 274mu3 respectively. The dry mass and volume of tetraploid nuclei was twice as big as that of diploid nuclei in both materials. The nuclear water content was neither significantly different between diploid and tetraploid nuclei nor significantly different between nuclei from controls and patients with hepatitis.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.