Characteristics of trophoblast cell reproduction in the connective zone of the rat placenta. II. Determination of the degree of ploidy of mitotic shapes

Morphological and cytophotometric studies have been made on polyploidization of placenta connective zone cells. Measurement of the DNA content in mitotic figures show that within a period of development ranging from day 13 to day 14 the bulk of mitoses (up to 25%) become tetraploid and octaploid. This may suggest that polyploidization of placenta connective zone cells proceeds via incomplete polyploidizing mitoses. Among tetraploid and octaploid mitotic figures, there are those corresponding to all the mitotic stages, from prophase to telophase. Consequently, mitosis in tetraploid and octaploid cells can reach telophase. In such cases polyploidization is likely to follow the acytokinetic mitotic pattern. A question of a certain maximum level of polyploidy that may be reached by cells due to the incomplete mitosis is discussed.     

Polyploidizing mitoses and the biological meaning of polyploidy in liver cells]

The ontogenetic polyploidization of hepatocytes is regarded, within which normal mitoses are changed to polyploidizing mitoses, and diploid hepatocytes transform into polyploid mono- and binuclear cells. A new hypothesis is put forward of the biological significance of the liver cell polyploidy. The hypothesis takes into account a high level of spontaneous chromosomal aberrations in mitotic hepatocytes. The chromosome structural changes interfere with mitosis resulting in the chromosomal imbalance. Polyploidy bestows for hepatocytes a tolerance towards a chromosomal imbalance. Some implications of the hypothesis are discussed: unbalanced genome of hepatocytes after the treatment with mutagens and mitotic stimulators; the reasons of liver cell polyploidy differences in mammalian species; mechanisms of radioresistance of hepatocytes. Chromosomal imbalance of polyploid hepatocytes is assumed to be the basis for wome chronic liver diseases in man.     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergent-trypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

Summary The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergenttrypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between the DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.In honour of Prof. P. van Duijn     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Biological significance of liver cell polyploidy: an hypothesis

The liver cell polyploidy phenomenon, a characteristic of many species of mammals, is reviewed. The liver parenchyma of adult animals represents a mixed population of mononuclear and binuclear cells with different number of chromosome sets and, therefore DNA content per nucleus. The polyploid hepatocytes are formed during postnatal liver growth as a result of a change from normal mitoses to polyploidizing ones. Hence, the polyploidization of hepatocytes is regarded as an equivalent of cell multiplication.An hypothesis of the biological significance of liver cell polyploidy is based on the fact of a high level of spontaneous chromosome aberrations in mitotic hepatocytes. Ploidy increase is known to give resistance against different kinds of genome alteration. Polyploidization of the liver cells ensures protection against deleterious consequences of the aberrant genome formation resulting from aberrant mitoses.Some implications of the hypothesis are discussed: the reasons for species-specific differences of liver cell polyploidy; the mechanisms of hepatocyte radioresistance; the relation of polyploidy to liver cell aging. The prerequisite factors for unbalanced cell genome formation are adduced: DNA and chromosome damage as the first step in the process, stimulation of mitosis as the second one. The aberrant polyploid genome of hepatocytes is assumed to be the cytogenetic basis for some chronic liver diseases in man.     

Age-Related Changes of Elements in the Coronary Arteries of Monkeys in Comparison with Those of Humans

To elucidate compositional changes of the coronary artery with aging, the authors investigated age-related changes of elements in the coronary arteries of rhesus and Japanese monkeys by direct chemical analysis in comparison with the coronary arteries of Japanese and Thai. Used monkeys consisted of 38 rhesus monkeys and 23 Japanese monkeys, ranging in age from newborn to 33 years. After perfusion with a fixative, the hearts were resected from the monkeys, and the anterior interventricular branches of the left coronary artery and the right coronary arteries were resected from the hearts. After ashing of the arteries, element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the Ca and P contents did not increase in both the left and right coronary arteries of rhesus and Japanese monkeys at old age. The average contents of Ca and P decreased by 13% and 25% in the left coronary arteries more than 20 years of age in comparison with those below 20 years of age, whereas they decreased by 4% and 15% in the right coronary arteries more than 20 years of age in comparison with those below 20 years of age. This finding indicated that atherosclerosis scarcely occurred in both the left and right coronary arteries of rhesus and Japanese monkeys at old age. In contrast with monkeys, atherosclerosis occurred frequently in the coronary arteries of Japanese and Thai at old age.     

Polyploidization of transplanted cardiac myocytes

Pieces of cardiac ventricular tissue of late embryonic or 1-day postnatal rats, implanted beneath the kidney capsule of adult syngeneic hosts, formed viable, beating transplants. these transplants were investigated over a 40-day postoperative course. In the transplants, cellular binucleation and nuclear polyploidization occurred according to the same schedule as in the heart in situ. The composition of the classes of myocytes was identical both in the hearts in situ and in transplants, but the number of non-diploid myocytes in the intact heart reached 90%, whereas in transplants it varied from 30 to 60%. In contrast to the heart in situ, myocytes in transplants grew feebly after the phase of polyploidization. From these data one can conclude that under conditions of transplantation the temporal sequence of cellular binucleation and nuclear polyploidization follows the normal course, but that a greater number of myocytes remain in a diploid state than is the case in the normal heart. The growth of cardiac myocytes seems to be related to their level of function.     

Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.     

Endopolyploidie im Endosperm vonEchinocystis lobata

Zusammenfassung Das Kernwachstum im Endosperm vonEchinocystis lobata geht auf endomitotische Polyploidisierung zurück. Besonders die Kerne des eigentlichen Endosperms erreichen hohe Endopolyploidiegrade, und zwar im zentralen Teil maximal 3072n, die des Chalazahaustoriums werden bis zu 24ploid. Die Struktur der meisten endopolyploiden Kerne, die keine postendomitotischen Mitosen durchgemacht haben, ist charakterisiert durch langgestreckte Endochromozentren, die sich nach jedem Endomitoseschritt vergrößern.Im chalazalen Teil des eigentlichen Endosperms laufen nach Abschluß der endomitotischen Polyploidisierung regelmäßig spontane, polyploide Mitosen ab; von dieser Mitosewelle werden triploide bis 48ploide und höchstwahrscheinlich auch noch 96- und 192 ploide Kerne erfaßt. Postmitotische polyploide Kerne zeigen bezeichnenderweise eine andere Struktur als endopolyploide, die keine Mitose durchlaufen haben, nämlich viele Nukleolen und zahlreiche kleine, schollige oder fädige Chromozentren.Im Nuzellus werden die Kerne endomitotisch schätzungsweise bis zu 16ploid.

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Summary The nuclei in the endosperm ofEchinocystis lobata show a considerable increase in size; this is due to endomitotic polyploidization. Particularly the nuclei of the endosperm proper attain high degrees of endopolyploidy: in the central parts the maximum is 3072n. The largest nuclei of the chalazal haustorium are 24n. Most of the nuclei which did not undergo postendomitotic mitoses have oblong endochromocentres enlarging after each endomitosis.In the chalazal part of the endosperm proper after endomitotic polyploidization regularly spontaneous mitoses take place. They are triploid as well as polyploid (6n to 48n and probably also 96n and 192n). Postmitotic polyploid nuclei show a structure different from that of endopolyploid nuclei which did not undergo mitoses, viz they contain many nucleoli and numerous small granular or filamentous chromocentres.The nuclei of the nucellus attain low degrees of endopolyploidy of approximately 16n.

     

Cardiac allograft pathology in Rhesus monkeys

Orthotopic, allogeneic heart transplantations were performed in 26 randomly selected rhesus monkeys to study the effect of immunosuppressive treatment on survival and cardiac morphology. The histological picture of the acute and chronically rejected hearts was similar to that described for acute and chronic cardiac allograft rejection in man. As seems to be the case in man, an obliterative arteritis of the coronary arteries appears to be the limiting factor in cardiac allograft survival in the rhesus monkey. Immunosuppressive treatment with either Imuran and ALS or Imuran, ALS, and Prednisone could not prevent the occurrence of obliterative arteritis of the coronary arteries. Contrary to the observation in man, no atherosclerotic changes were found in the affected parts of the coronary arteries or in the non-diseased parts.     

Somatic polyploidy in neurons of the gastropod mollusca. III. Mitosis and endomitosis in the postnatal development of neurons in the Succinea snail central nervous system

General morphology of chromatin, the number of chromosomes and chromocenters in normal condition and at the increase of bivalent cation (Ca2+, Mg2+) concentration were studied with the purpose to reveal mechanisms of polyploidization of neuron nuclei in the snail Succinea lauta (Gastropoda, Pulmonata). The morphology of nuclei was studied on squashed preparations. Normal diploid mitoses are described in the cerebral ganglia. A possibility is supposed that part of neurons or neuroblasts in the central nervous system (CNS) of succineid snail may divide mitotically. It has been shown that the basic mechanism of neuron postnatal growth is endomitotic polyploidization of nuclei. The transition from ordinary mitosis to polyploid cycles occurs via restitutional (polyploidizing) mitosis (4c2n-->4c4n). The next endocycles are carried out by means of classic endomitosis up to reaching the highest ploidy levels--4096n--16,384n. The study of general morphology of chromatin and chromocenters at normal condition and at artificial compactization enabled us to exclude any probability of polyteny in the CNS of lauta.     

Cytofluorometric nuclear DNA determinations on the atrioventricular nodal cells in human hearts

In the human heart, it is well known that the polyploidization of working heart-muscle cells increases in proportion to increases in heart weight, but there has been no investigation of the process of polyploidization in the specialized heart-muscle cells of the cardiac conduction system which have a nerve-like function. In order to investigate the process of polyploidization in these cells, the nuclear DNA content of atrioventricular nodal cells was measured using cytofluorometry. Tissue samples taken from autopsied hearts without arrhythmias were embedded in paraffin blocks after Carnoy fixation. Blocks containing the atrioventricular conduction system were cut according to the serial sectioning method of Lev et al. The compact atrioventricular nodes were removed from thick paraffin sections (150 micron) under a stereomicroscope. The cells were then isolated by enzyme digestion and ultrasonic treatment. Smears of the isolated cells were double stained with azocarmin-G and acriflavine-Feulgen. Cytofluorometric DNA determinations of the DNA content of atrioventricular nodal cells were performed. Atrioventricular nodes were found to be composed of a large number of diploid cells and a small number of tetraploid cells. No octaploid cells were found. These findings reveal that the process of polyploidization in atrioventricular nodal cells is different from that found in working heart-muscle cells.     

Genome multiplication in the trophoblasts and glandular epithelium of endometrium during embryo implantation and placentation of silver fox

Dynamics of genome multiplication during establishment of interrelations between the trophoblast and the glandular epithelium of endometrium was studied in the course of placenta formation in the silver fox. Endometrium response on the embryo implantation exhibits some features of inflammation. In the course of placenta formation the trophoblast gains access to the endometrial glandular epithelium zone, while the endometrial blood vessels grow the other way into the expanding trophoblast zone. The trophoblast gradually replaces the whole epithelium and part of the stroma of the endometrium, closely adjoining the endometrial vessels but not disrupting them. Cytophometric DNA measurements in the trophoblast nuclei have shown that most of the nuclei are polyploid: predominantly 4c-64c, occasionally 128c and 256c. Polyploidy of the trophoblast may result from various types of polyploidizing mitoses. Cytophotometric DNA measurements in mitotic figures have revealed mitoses with DNA amounts equal to 4c (2n), 8c (4n), and 16c (8n), which indicates that trophoblast cells in the silver fox placenta are able to enter mitosis prior to the octaploid level. Higher degrees of polyploidy in the trophoblast cells may be achieved presumably by endoreduplication. In the silver fox polyploidization of uterine grandular epithelial cells during placentation occurs until the level of 8c. Thus, the tissue-specific response of the uterus to the implanting embryo is an active proliferation and polyploidization of the glandular epithelium, rather than formation of a population of polyploid decidual cells (i.e. connective tissue cells). Using the silver fox endotheliochorial placenta as an example, a regularity has been confirmed that cells of both maternal and fetal origin are polyploid in sites of their contact in placenta, which might be of protective significance in the contact of allogenic organisms.     

Ploidies of cardiomyocytes in human myocardial hypertrophy]

DNA cytophotometry has been performed in ventricular cardiomyocytes of hypertrophic human hearts. In the cases of hypertrophy in adults (generalized atherosclerosis, postinfarct scars), polyploidy expression did not exceed the limits of normal variability developed during childhood. In the cases of hypertrophy caused by congenital heart defects, high polyploidy has been revealed (the mean level 20c and more, where c is haploid DNA content), which considerably exceeded the upper limit of normal variability (approximately 10c). Our hypothesis has confirmed that heart hypertrophy in adults proceeds in conditions of stable genome rather than due to redundant polyploidization of the ventricular myocytes. The same idea assumes enhanced polyploidization of the myocytes in childhood in humans with congenital heart diseases.     

Polyploidy: significance for cardiomyocyte function and heart aerobic capacity

Somatic polyploidy, defined as genome multiplication, was found in all differentiated mammalian tissues. The highest level of such a polyploidy was found in the myocardium. This phenomenon was shown to be associated with changes in the pattern of gene expression. Hence, polyploidization may create cells with new physiology. The effect of polyploidy on the heart function has never been studied. The aim of the present study was to investigate the effect of polyploidy on cardiomyocyte functioning and heart aerobic capacity. DNA and the total protein content, nucleolar activity reflecting the rate of rRNA synthesis and, consequently, ribosome biogenesis, were measured in ventricular myocytes isolated from the human and from 21 mammalian species by image cytometry and microscopic morphometry. The total protein content was estimated after staining slides with naphtol-yellow dye. For measurement of DNA and nucleolar area, staining with Hoechst and AgNO3 was applied. Cardiac aerobic capacity was evaluated by the heart mass to body mass ratio. A negative correlation between the heart index and the average cell ploidy was revealed (r = -0.79; P < 0.0001). The average genome number per myocyte was registered to be higher by approximately 35% in the sedentary mammals, with the heart index about 0.4% from body mass, than in the athletes with heart index about 0.6% of body mass. Polyploidization was shown to be associated with a sharp decrease in the protein/DNA ratio in cardiomyocytes. As a result, cardiomyocytes in the athletic mammals with poorly polyploid hearts have much higher protein content per genome than do cells in the sedentary species with highly polyploid hearts. Surprisingly, despite decreased protein/DNA ratio, the nucleolar area per genome significantly increased with polyploidization, indicating the imbalance between the cellular protein content and the rate of ribosome biogenesis. Such an imbalance should obviously impair cardiac function, because the additional genomes take some valuable space and biological resources from the cell, which could have been otherwise directed to the maintenance of cardiomyocyte contractile machinery. It is generally accepted that somatic polyploidy is associated with oxidative stress and energetic starvation. Thus, we suppose that additional genomes may serve for cardiomyocyte protection from oxidative damage in the hearts.     

Mitoses and binucleated cells in perinatal human hearts

Cells from hardened formalin-fixed human hearts were isolated with potash lye. In perinatal hearts mitoses and evidence of irregular chromosomal movement such as chromatin bridges and micronuclei were observed in small numbers during normal development. A quantitative analysis of binucleated cells shows 8.8 +/- 5.3% in the left and 11.7 +/- 4.0% in the right ventricular wall around the time of birth. The number increases during the first year of life to 56.8 +/- 17.4% in the left and 42.0 +/- 18.1% in the right heart. This development precedes normal polyploidisation of the human heart by years.     

A method for investigating hepatocyte polyploidization kinetics during postnatal development in mammals.

A method for investigating weakly-proliferating cell populations of liver parenchyma on the basis of a quantitative analysis of hepatocyte polyploidization during postnatal development is described. The method uses a mathematical model which characterizes the hepatocyte polyploidization process, and incorporates data concerning the time course for relative frequencies of hepatocytes in different ploidy classes. As a result of these measurements and calculations for rat liver, transition rates of hepatocytes (the relative number of cells during a given time unit) from one ploidy class to another, and a coefficient for the reduction of hepatocyte mitotic activity with an increase in its ploidy class were obtained. Calculated curves show a good correspondence with the real process of hepatocyte frequency changes as they relate to changes in the age of the animals. To check this method, experiments investigating time changes of autoradiographic label content in the different ploidy classes of hepatocytes were carried out. By mathematically modeling the label diluting process resulting from cell proliferation and polyploidization, transition rates of hepatocytes were calculated, and they reflect values calculated from the model according to changes in occurrence frequencies.     

Cytofluorometric nuclear DNA determinations on the atrioventricular nodal cells in human hearts

Summary In the human heart, it is well known that the polyploidization of working heart-muscle cells increases in proportion to increases in heart weight, but there has been no investigation of the process of polyploidization in the specialized heart-muscle cells of the cardiac conduction system which have a nerve-like function. In order to investigate the process of polyploidization in these cells, the nuclear DNA content of atrioventricular nodal cells was measured using cytofluorometry. Tissue samples taken from autopsied hearts without arrhythmias were embedded in paraffin blocks after Carnoy fixation. Blocks containing the atrioventricular conduction system were cut according to the serial sectioning method of Lev et al. The compact atrioventricular nodes were removed from thick paraffin sections (150 m) under a stereomicroscope. The cells were then isolated by enzyme digestion and ultrasonic treatment. Smears of the isolated cells were double stained with azocarmin-G and acriflavine-Feulgen. Cytofluorometric DNA determinations of the DNA content of atrioventricular nodal cells were performed. Atrioventricular nodes were found to be composed of a large number of diploid cells and a small number of tetraploid cells. No octaploid cells were found. These findings reveal that the process of polyploidization in atrioventricular nodal cells is different from that found in working heart-muscle cells.