Ploidy Classes Research Articles

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.

Megakaryocytes and the heterogeneity of circulating platelets.

Megakaryocytes mature to the point of cytoplasmic disintergration in three principal ploidy classes: 8n, 16n and 32n. Cells of each of these ploidy classes have been identified, using both microdensitometry and measurement of cell volume and submitted to morphometric analysis. Mature megakaryocytes of the three ploidy classes have been shown to differ in the concentration of cytoplasmic constituents which would be expected to relate to the buoyant density of their platelet progeny. Density separated platelets have been similarly analysed. Light platelets correspond with the 32n megakarycytes and are more liberally endowed with surface connected canalicular system than the progeny of the common 16n megakaryocytes; it is proposed that they have functional characteristics related to this finding. Dense platelets, which are larger in size, correspond with 8n megakaryocytes and show a greater content of granules and mitochondria than platelets of average density. These platelets most probably show specialized function relating to release of granule constituents. Fragments of cytoplasm released from megakaryocytes represent one form of "megathrombocyte" equated by others with newly formed platelets. The differences in structure between these fragments and circulating platelets are emphasized; each such fragment must undergo further disintergration into a number of platelets. It is suggested that the heterogeneity of circulating platelets with respect to both size and density stems from the origin of platelets of varying density from the different populations of megakaryocyte and their release in the form of large cytoplasmic fragments rather than as platelets.

Ultramicrochemical determination of nucleic acids in individual cells using the Zeiss UMSP-I microspectrophotometer. Application to isolated rat hepatocytes of different ploidy classes.

Edström's method for the ultramicrochemical determination of RNA and DNA in individual cells was modified for the measurement of extinction in u.v. light with the aid of the Zeiss scanning microspectrophotometer UMSP-I. With this new procedure, nucleic acids down to about 3 pg RNA or about 4 pg DNA can be measured with a very high accuracy. The method was applied to enzymatically isolated rat liver parenchymal cells. A mean DNA content of 6.52 pg was found for diploid cells. The DNA content of mononuclear cells of different ploidy levels and of binuclear cells showed a close proportionality with the nuclear ploidy and the number of nuclei per cell. The RNA content of mononuclear diploid cells amounted to 33.4 pg, yielding an RNA/DNA ratio of 5.12. The RNA/DNA ratio was similar for binuclear and mononuclear cells of the same ploidy level but decreased considerable with increasing nuclear ploidy.

Platelet density and size: the interpretation of heterogeneity.

Platelets have been separated according to buoyant density using a colloidal silica-polyvinylpyrrolidone system and subjected to electronic sizing. All density populations were found to be heterogeneous in size, the most dense platelets ranging from less than 3 fl to greater than 21 fl in both man and rat. Light platelet fractions contained no platelets greater than 13 fl in either species. Cohort labeling with [75Se]selenomethionine showed no indication of significant change in platelet buoyant density with ageing; greater specific activity found in young, dense platelets appears to be related to increased protein synthetic activity shown in vitro and likely to occur also in their precursor megakaryocytes. It is postulated that dense, intermediate and light platelets are released synchronously by the three different ploidy classes of megakaryocyte, that varying density indicates differing structural characteristics and presumably differences in function. The present findings do not deny the possibility that platelets decrease in size with ageing but if such occurs, it is not associated with a significant change in platelet buoyant density.

Chloroplast replication and chloroplast DNA synthesis in spinach leaves

Cultured leaf disks from young spinach leaves have been used to study chloroplast DNA synthesis in relation to chloroplast growth and replication. Autoradiographs prepared from thin sections show that labelling throughout disks is uniform. We consider that cultured leaf disks serve as a model system for studies of cell expansion and chloroplast replication during the development of spinach leaf cells. Leaf disks from young spinach leaves were incubated continuously for 5 days in a medium containing [ 3 H]thymidine. They were harvested daily and light microscope autoradiographs of separated cells prepared. In the expanding cells of disks all chloroplasts divide and the synthesis of chloroplast DNA as measured from grain counts was correlated with the rate of chloroplast formation. A doubling of chloroplast numbers was associated with a doubling of chloroplast silver grains, which is consistent with a duplication of chloroplast DNA during the chloroplast division cycle. When disks were grown in green light, chloroplast replication but not chloroplast growth ceased in 3–4 days. The large chloroplasts of disks grown in green light appeared locked in a dumb-bell shaped configuration. Light microscope autoradiographs have established that these chloroplasts can synthesize DNA. These chloroplasts divided when the disks were exposed to high intensity white light and again it appears that all chloroplasts in the one cell divide. The chloroplasts of disks grown in green light for periods up to 9 days became uneven in size although all were dumb-bell shaped. We suggest that the size differences represent ploidy classes. Chloroplast replication in dicotyledons has been discussed in relation to the division and growth of leaf cells.

Time Course of Polyploidisation in Calli Derived from Stem and Pith Explants of Nicotiana tabacum studied on Isolated Nuclei

Summary A DNA cytophotometric analysis was performed on interphase nuclei isolated from pith and stem of Nicotiana tabacum and calli derived from these tissues during a six months period of in vitro culture. The results showed that the pith is made up of a cell population which is essentially polyploid, whilst the great majority of stem nuclei has diploid DNA content. At the end of the experimental period, regardless of the nuclear DNA contents in the expiants, the frequency distribution of the various classes of ploidy is very similar in both types of calli. There is an increase in frequency of 4 C nuclei, which become with time the predominant class. Thus the in vitro conditions of the present experiment caused in our material a reduction of ploidy in the calli obtained from pith and a slight increase in ploidy in the calli derived from stem.

“Non-condensed” and “condensed” chromatin 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.

Age-dependent ploidy class changes in mouse hepatocyte nuclei as revealed by Feulgen-DNA cytofluorometry

The polyploidization of hepatocyte nuclei was quantitatively studied, without regard to binuclearity, by the Feulgen-DNA cytofluorometric method with smear preparations from male C57BL/6 mice of 0·03-, 0·33-, 1-, 2·5-, 4·0-, 4·5-, 8-, 14-, 21-, 26·5- and 28-months-old. About 300 nuclei per liver were measured on their DNA contents and classified into various ploidy classes using the DNA content of small granular cell nuclei of the cerebellum as a standard, i.e. the diploid (2C). The percentage of 2C nuclei was 99 per cent at 0·03-month, but rapidly decreased with age and finally fell to 8·5 per cent at 28-months. The 4C fraction was 1 per cent at 0·03 month and began to increase to a maximum of 59 per cent at 4·5-month followed by a gradual decrease to 23 per cent at 28-months. The 8C nuclei was first detected at 1 month (0·8 per cent) and the percentage steadily increased to 45 per cent at 28-months. The highest ploidy class observed was 32C at 28-months (4 per cent). By expressing these results in terms of Polyploidization Index (P.I.), i.e. percentage of polyploid nuclei divided by percentage of diploid nuclei, then the following relationship was found: P.I. = 0·393 X Age (month) 0.881. Furthermore the correlation coefficient between logarithm of P.I. and logarithm of age in months was found to be +0·892.

DNS in Kinderherzen. Biochemische und zytophotometrische Untersuchungen

In order to clarify the question of an age-dependent polyploidisation of the myocardium during normal cardiac growth, the DNA-content of 39 hearts of infants and children was investigated. After autopsy the total cardiac weights were estimated and thereafter, following the preparation of the hearts, the pure weight of the myocardium (preparation weight) was determined. Using the diphenylamine reaction according to Dische and Burton, the DNA-concentrations and total amounts of DNA in the heart muscle were estimated. The DNA-content of the heart muscle cell nuclei (ploidy classes) was measured cytophotometrically on Feulgen-stained smears of heart muscle cells. DNA measurements were performed on tissue samples from 6–8 different sites of both heart chambers. Growing human hearts of all ages were investigated including 3 fetal hearts and 3 hearts with malformations. The investigations showed the following results: 1. During fetal cardiac growth the biochemically estimated DNA-concentration increases rapidly and reaches the highest values just after birth. In the first years of life the DNA-concentration decreases to 400 µg/g tissue which is found between the 8th and 12th year of life when the pure myocardium weighs 100 g. This DNA-concentration is the same in hearts of older children and of adults. 2. The total amount of myocardial DNA increases in growing hearts up to adult age concomitantly with the increasing heart weight. After birth the DNA-content of the heart is 20 mg, in normal adult hearts it is 100 mg. 3. The DNA-amount as estimated from 300 heart muscle cell nuclei is 1.914 × 10 −9 g in hearts of infants; it is about twice this value (3.89 × 10 −9 g) in hearts of older children. 4. From birth to the age of 7 years 80 to 90% of the cell nuclei are diploid, with only 7 to 16% tetraploid nuclei. At the age of 7 years a polyploidisation begins with an augmentation of tetraploid muscle cell nuclei up to 42% and a decrease of diploid nuclei to 56%. In the heart of an 8-year-old child there are only 30% diploid but 58% tetraploid cell nuclei; 4% of the nuclei are octoploid. 5. In the right ventricle the main process of polyploidisation takes place 4 years later (in the 12th year of life) than in the left ventricle. There are 56% diploid and 26% tetraploid cell nuclei in the right ventricle of an 11-year-old child and, on the other hand, only 22% diploid and 56% tetraploid nuclei in the right ventricle of a 12-year-old. The muscle cell nuclei of the left ventricle, however, are already polyploid at the age of 7. 6. In fetal hearts 60% of the cell nuclei are aneuploid (3c) with a broad scattering of the data measured indicating the presence of cells in the S-phase. The tetraploid DNA-values within these hearts relate probably to premitotic cell nuclei. 7. In hearts with malformations a strong shift of the ploidy niveau towards higher DNA-values occurs; the polyploidisation is much more marked in the hypertrophied right ventricle than in the left one. Some sites of the left ventricle showed a DNA-distribution according to age. In the heart of a 3-week-old child with atrial septal defect a DNA-distribution pattern according to age was found which points to a time factor for the polyploidisation. The physiological polyploidisation of the human heart muscle in childhood is correlated with the general growth process. An increased hyperfunction as a stimulus for DNA-synthesis is discussed; a hormonal stimulus, on the other hand, cannot be excluded.

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.

Cell kinetics of mouse urinary bladder epithelium. I. Circadian and age variations in cell proliferation and nuclear DNA content.

The urinary bladder epithelium contains nuclei with diploid, tetraploid and octoploid DNA content. A study in mice has been performed on mitotic activity, DNA synthesis and ploidy. The study has confirmed previous findings of an extremely low mitotic activity and a LI of 0.40 +/- 0.04%. The results indicate a higher DNA synthesis during the night as compared to the day. The relative numbers of diploid-, tetraploid- and octoploid cells have been estimated by micro-flow fluorometry. The mean values of these different classes were diploid: 39.40 +/- 1.4%, tetraploid: 53.27 +/- 1.00% and octoploid: 4.41 +/- 0.22%. Hexaploid DNA values with a mean of 2.51 +/- 0.13% were found and the significance of hexaploid pulses is discussed. The different ploidy classes changed during the neonatal period with a decrease of the diploid class and an increase of the tetraploid class. A reduction of the octoploid class about 4 weeks after birth was seen. The distribution of the different classes became stabilized around the second month.

The recognition and incidence of haploid and polyploid spermatozoa in man, rabbit and mouse.

The existence of polyploid mammalian spermatozoa has been inferred from studies of Feulgen-DNA absorption. Rabbit spermatozoa fell into two discrete groups with mean absorptions close to a 1:2 ratio (inferred to be haploids and diploids respectively); simple visual appraisal of the size of the head or nucleus gave an identical classification. The incidences of ploidy classes were 98-94% haploid, 1-06% diploid, 0-00% higher than diploid (N = 3010; from DNA measurements and visual appraisal of the size in a rabbit chosen to have a high incidence of diploids) and, correspondingly, 99-691%, 0-308%, 0-001% (N = 138001; from sixty-nine unselected rabbits, scored by visual appraisal of the size of the sperm head). In man also, virtually discrete groups with absorptions close to a 1:2 ratio existed and were inferred to be haploids and diploids respectively. A few human spermatozoa were found with absorptions corresponding to a ploidy of three and/or four. Visual appraisal of the size of the human sperm nucleus as Small, Medium or Large was only a partial guide to ploidy. All Small human spermatozoa measured for DNA absorption were found to be haploid. About two-thirds of Medium human spermatozoa were found, however, to be haploid, and some Large spermatozoa were haploid or diploid. The incidences of ploidy classes in the human were 99-37% haploid, 0-56% diploid, 0-07% higher than diploid (N = 5554; with consistency between duplicate slides and between two subjects; from DNA measurements and visual appraisal of nuclear size). The estimated incidence of diploid human spermatozoa is consistent with the known incidence oftriploid fetuses. In a mouse with a putatively high incidence of diploids, all 1000 DNA measurements were nevertheless within the haploid range, with one diploid encountered outside the main sampling.

An improved method for the separation of cells by sedimentation at unit gravity

An apparatus is described for the separation of cells by sedimentation velocity at l g. The viscosity of the sample was raised by the addition of polyethylene oxide and subsequently the sample was layered on top of a density gradient via a sieve. With the aid of this procedure the various ploidy classes of rat-liver cells were enriched and murine leukemia cells were separated according to the phases of their life cycle.

The DNA content of megakaryocytes from the bone marrow and vessels of the lung in rats.

Comparative microspectrophotometry of the DNA content of Feulgen-stained megakarocytes from the bone marrow and from those retained in the pulmonary circulation showed that the ploidy classes 8 N, 16 N and 32 N are found in both bone marrow and pulmonary circulation, whereas the 64 N class is found in the bone marrow ocal 'naked nuclei'.

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.

Zellklassen und Ploidiestufen in isolierten Leberzellen der Maus

Hepatocytes isolated from the liver of 5-month-old male NMRI-mice were used to determine the frequency of mono-, bi-, and multinucleate cells. Ploidy distributions were measured by karyometry and Feulgen-cytofluorometry. 75% of the mouse liver cells are binuclear, mainly with 4N or 8N nuclei. 15% of all nuclei belong to higher ploidy classes (16N–128N).

Some quantitative data regarding the nucleoli in cell nuclei from rat liver of different ploidy classes.

In an investigation comprising different morphometric and numerical data with regard to the nucleoli in smears of isolated parenchymal nuclei of rat liver cells, the following facts were established. 1. Within the three different ploidy classes existing in adult animals, no correlation has been observed between the nucleolar projection area and the number of nucleoli per nucleus. A considerable overlap exists between the numbers of nucleoli in the diploid, tetraploid and octoploid class, so that it is not possible to classify the nuclei of the three classes on the number of nucleoli. 2. The total nucleolar projection area per nucleus in a given ploidy class varies within rather narrow limits, independently of highly variable numbers of individual nucleoli. The means of these nucleolar areas in diploid, tetraploid and octoploid nuclei, showed virtually no deviation from an 1∶2∶4 ratio. 3. The mean numbers of nucleoli in the livers of young adult animals (8 weeks of age) are 2.37, 5.02 and 10.08 for the 2n, 4n and 8n classes, respectively. In embryos and newborn animals, the modal number of nucleoli is 4 in the diploid nucleus, while the first appearing tetraploid and octoploid nuclei, later in development, show at first 8 and 16 nucleoli respectively, the mode of which number falls quickly afterwards. The reduction in nucleolar number, undoubtedly due to fusion, proceeds continuously up to old age: in rats of 21 months, the means are reduced to 1.45 (2n class), 2.66 (4n class) and 5.85 (8n class).

Ventricular nuclei-DNA relationships with myocardial growth and hypertrophy in the rat.

Rat ventricles, ranging from 306 to 1999 mg, were obtained from normal animals of different ages and from animals subjected to chronic aortic constriction. The concentration of DNA in the paired ventricles (right and left) was found to be closely related to the concentration of nuclei in the left ventricular papillary muscles. Muscle nuclei represented only 10 to 15% of this population of nuclei. In young animals (phase 1), the total ventricular content of muscle nuclei, nonmuscle nuclei, and of DNA were increasing with ventricular growth. In the adult rat (phase 2), the total ventricular content of DNA and of both muscle and nonmuscle nuclei remained relatively constant with ventricular growth. In the enlarged hearts (phase 3), there was a further increase in total ventricular DNA but there was no further increase in the total number of muscle nuclei. Spectrophotometric studies (Feulgen stain), showed that 88% of the muscle nuclei belonged to a single ploidy class (probably diploid). No relation could be demonstrated between the extent of nuclear polyploidy and the weight of the ventricles. It was concluded that polyploidy was not a significant factor in the increased total DNA of phase 3. The increased ventricular DNA of phase 3 was explained by the proliferation of the nonmuscle nuclei.

VIII. Untersuchungen zum Kohlenstoff-Stoffwechsel unbestrahlter Zellen

Some quantitative data about the carbon-metabolism in Saccharomyces-cells of different ploidy were determined. The amount of carbon, necessary for the formation of a cell, proved to be proportional to the degree of ploidy of the cells. For the duplication of a diploid cell 6,7·10-11g glucose were used. In comparison with respiratory deficient cells the simultaneous utilization of fermentation and respiration metabolism in respiration sufficient cells leads to a decrease of the cell cycle duration, however, the energy needed for the formation of a cell is not decreased. The rate of cell multiplication has a maximum at about 30 °C for all classes of ploidy. Certain assumptions about the utilization of the carbon source were confirmed by experiments with 14C marked glucose.

Further studies on the replication of rat liver cells in vivo

The study of the replicating cells of the liver at different ages from 1 day to 36 months shows that the diploid cell is the chief multiplying unit. The data indicate that the replication patterns and times of the diploid cell at 3 and 8 weeks and 6 months of age are similar. At 1 day, its replication time is shortened, but its DNA synthesis time is similar to that of diploids at other ages. At 8 weeks of age a few tetraploid liver cells replicate. A significant change during growth is the decreasing size of the replicating pool of cells. The method of studying the replication of cells through the changes in their ploidy classes is useful in analyzing asymmetrical mitotic labeling curves with more than one replicating ploidy class, and in instances where too few mitoses are present for the determination of mitotic labeling curves.