Changes in the amount of 3H-thymidine label in hepatocytes of different ploidies in rat ontogeny after a single administration of the isotope]

Change of 3H-thymidine quantity in mono- and binuclear rat hepatocytes of different ploidy was investigated during the first 6 weeks after a single injection of isotope to newborn rats. Rates of cell transitions (arbitrary number of cells in the time unit) from one ploidy class to another, and coefficients of the reducing of hepatocyte proliferative activity with increasing the hepatocyte ploidy were calculated on the basis of ideas about the process of autoradiographic label "diluting" in the course of the postnatal development as a result of polyploidization and ordinary mitotic divisions of hepatocytes. The calculated values are close to values of parameters, which were calculated with assistance of the model, which describes the process of polyploidization in the liver, on the basis of data on the change in the arbitrary number of different ploidy hepatocytes.     

Peculiarities of liver regeneration in Chinese hamster <Emphasis Type="Italic">Cricetulus griseus</Emphasis>

With the aid of cytofluorimetry and interference microscopy, the ploidy level and the hepatocyte ploidy class distribution were studied and the dry mass of hepatocytes was measured in hepatocytes in liver of Chinese hamsters Cricetulus griseus and of Balb/c mice before and one month after partial hepatectomy. The mean ploidy level in hepatocytes of the Chinese hamster normal liver amounted to 2.35 ± 0.03 c. The modal class was mononuclear hepatocytes with diploid nuclei (82.4 ± 1.3%). The mean dry mass of hepatocytes amounted to 605.2 ± 4.8 pg. In the process of liver regeneration in the Chinese hamsters, the ratio of ploidy classes and the hepatocyte dry mass did not change. After a similar liver resection in the mice, a significant polyploidization of liver parenchyma occurred. The mean ploidy level in hepatocytes rose by 32%. Instead of 4cx2-hepatocytes, the modal class became mononuclear octaploid cells the relative portion of which increased, on average, by five times. The portion of binuclear hepatocytes with octaploid nuclei in mouse liver rose by more than five times. Thus, in the Chinese hamsters Cricetulus griseus, unlike mice, regeneration of liver occurred exclusively at the expense of proliferation of hepatocytes.     

Analysis of the polyploidization kinetics of the parenchymal cells in the rat liver

Methodological approaches to kinetics of cell polyploidization in the rat liver parenchyma are discussed. Different ways of hepatocyte polyploidization in the course of postnatal liver growth have been assessed. The intensities of hepatocyte transitions from one ploidy class to another were determined. On the basis of literary experimental data the following is summarized: With the increase in the animal age, there is a decrease in hepatocyte transition from one ploidy class to and ther; in young animals the intensity of formation of tetraploid hepatocytes through the stage of binuclear cells (2c----2c X 2----4c) is 0.39-0.55 within two weeks, the intensity of direct transitions (2c----4c) being 0.00-0.19 within the same time. The intensity of entering to DNA synthesis is reduced with the increase in hepatocyte ploidy levels; in this case the coefficient of the reducing of mitotic activity is calculated as 0.10-0.22, and 0.01-0.05 for 4c- and 8c-hepatocytes, resp. The factors stimulating proliferation in the liver increase the intensity of the direct cell transition (2c----4c) by several times which can exceed the intensity of transition through the binuclear cell stage.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Regeneration of the liver of Chinese hamster Cricetulus griseus

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.     

The kinetics of the cell population of human liver parenchyma at different periods of life]

Processes of polyploidization in the liver parenchyma were investigated in the course of postnatal organism growth, stabilization of growth and ageing, using cytophotometry on the slides of isolated hepatocytes from normal livers of 140 donors aged from 1 day to 92 years. In addition, livers of human embryos (4, 5, 6 and 7 month old) were investigated. It is concluded that polyploid cells in the human liver appear in individuals aged from 1 to 5 years. However, during the postnatal development their relative number increases insignificantly. At the end of the intensive postnatal growth period the share of polyploid human liver cells is less than 3%. Binuclear cells with diploid nuclei are seen as early as in the embryonic liver. After birth their number increases slowly to reach 7.1% in the 16-20 year age group. The postnatal growth of human liver is due mainly to mitotic divisions of mononuclear diploid hepatocytes whose relative number is more than 90% during the postnatal growth. During the period of maturity (from 21 to 50 years), when the liver practically stops to grow, the levels of hepatocyte ploidy are changed insignificantly: part of 2c-hepatocytes decreases slowly (up to 84.8% by the end of period) and (2c x 2)-hepatocyte number increases slowly too. The number of polyploid cells increases by several times, but is equal only to 6.6% of all the hepatocytes counted. Under ageing, on the background of human liver atrophy, acceleration of hepatocyte polyploidization takes place. In the age group of 86-92 years parts of 2c- and (2c x 2)-hepatocytes reach 60.3 and 14.3%, resp., and the total share of polyploid cells is as much as near 25%, calculated from the cell population of liver parenchyma. The maximum ploidy levels in hepatocytes of normal human liver during ageing is becoming 16c and 8c x 2 for mononuclear and binuclear cells, resp. Transition rates among hepatocytes of different ploidy classes (2c--2c, 2c--2c x 2, 2c x 2--4c, 2c--4c) were calculated in addition to the coefficient of changing of the hepatocyte proliferative activity with the increase in its ploidy and cell death rate in different periods of human life. A rather high hepatocyte proliferative activity in the early postnatal period of human life was seen to lower during the following years of life. In maturity it is the lowermost to make less than 5% of that in newborns. During ageing the hepatocyte DNA-synthesizing activity being almost 1.6-1.7 times as much as in maturity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Progressive polyploidy in mouse liver following repeated hepatectomy]

Mouse liver regeneration after partial hepatectomy results in sharp changes of ploidy classes towards the increase of high ploidy cells and the decrease low ploidy ones. These changes retain during three months. Each following partial hepatectomy (till 3 times) intensifies the hepatocyte polyploidy with appearance of cells with 32--64 ploidy nuclei. The cell polyploidization stimulated by repeated regenerations is similar to that observed in normal postnatal liver growth.     

Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.     

Analysis of the postnatal growth of the mouse liver by estimating the hepatocyte count, their weight and ploidy

Changes in the total number of hepatocytes, their distribution by the ploidy classes, as well as changes in the protein content of the cells were studied in 0.5-6 month old mice. The data obtained made it possible to estimate quantitatively the contribution of different growth components: increase in cell number, hypertrophy and polyploidization of cells, to the total increase of the liver mass. From 2 weeks to 1 month, the liver mass is increased via polyploidization (by 70%) and hypertrophy (by 30%). From 1 to 2 months, the liver mass increases due to hyperplasia (by 65%) and polyploidization (35%). After 2 months, the liver growth is practically terminated. The calculated equivalent mass of the liver, i. e. derivative of all three growth components, coincides fairly well with the factual changes in the liver mass.     

Morphofunctional changes in hepatocytes during the early postnatal development of rats experimentally infected with the intestinal protozoan pathogen,Cryptosporidium parvum (Coccidia,Sporozoa)

Morphofunctional changes in hepatocytes of 10-14-day old rats were followed in norm and after experimental infection with different doses of oocysts of Cryptosporidium parvum. The liver index (ratio between the liver and body masses) varied with the intensity of invasion on the background of slowing down up to the total cessation of animal growth rates, and all this obviously pointed to severe pathology. In the infected rats, some cytological indices were shifted compared to the norm: protein amount and the average number of genomes per hepatocyte were seen to increase, the normal ratio between cells with different ploidy levels being violated. The particular correlation analysis was employed to distinguish between the ontogenetic (animal growth related) and pathologic (related to the infection intensity) polyploidization and hypertrophy in hepatocytes. In 10-14-day old rats, the former is affected primarily by the increase in the share of multinuclear hepatocytes, whereas the latter is accomplished by the increase in the number of cells with polyploid nuclei (4c and 4c x 2 cells). In the heavily infected rats, the ontogenetic polyploidy was almost totally suppressed due, presumably, to their growth rate inhibition, the rise in hepatocyte ploidy resulting form the obvious pathological changes in the liver. In the infected rats, the ontogenetic hypertrophy of hepatic parenchymatous cells was not manifested, and the observed protein accumulation in hepatocytes also resulted from the pathological changes in the liver. It is obvious that changes in cell hypertrophy (protein content) may serve as a more susceptible tool that readily perceives the host's stress experienced due to the parasitic infection (cryptosporidiosis), than cell ploidy: the levels of the respective responses of these two parameters differing by 4 times. However, due to the known reversible nature of hypertrophy, it cannot be used for the aims of a long-term prediction about the future mode of liver functioning in the animal that survived cryptosporidiosis. Unlike, such a parameter as frequencies of hepatocytes with different ploidy levels is much more useful in this respect.     

Whole-body X-irradiation of mice accelerates polyploidization of hepatocytes

Male C57BL/6 mice were whole-body irradiated with 4.75 Gy of X-rays at the age of 2 months and killed at 2, 6, 12 and 19 months after irradiation. The percentage survival began to decline earlier and faster in the irradiated group than the controls up to 19 months after exposure when the study was terminated. The nuclear DNA content of individual hepatocytes was measured by a Feulgen-DNA microfluorometric method, and hepatocytes were classified into various ploidy classes. In the irradiated mice, the degree of polyploidization was significantly higher than the controls by 2 months after exposure and steadily increased up to 6 months after exposure. Thereafter, however, a slow return to the control level was found up to 19 months after irradiation. These results appear to support a hypothesis that radiation accelerates the ageing process as judged from hepatocyte polyploidization.     

Ploidy-Dependent Growth and Binucleation in Cultured Rat Hepatocytes

The proliferative activity of rat hepatocytes, cultured in the presence of epidermal growth factor (EGF) and insulin, was examined by immunostaining of S-phase cells labeled with bromodeoxyuridine (BrdU) in culture. Proliferation rates of the different hepatocellular ploidy and nuclearity classes were measured by fluorescence image cytometry or by microscope counting of immunostained cells. Effects of EGF and insulin were largely additive, the binuclear cells being more growth factor-dependent (showing less growth in the absence of factors) than the mononuclear cells. A serial warm-washing procedure was used to remove excess BrdU from the culture medium, allowing the study of hepatocellular binucleation by a BrdU pulse-chase approach. A high rate of binucleation was detected (50%, possibly suggesting a quantal mechanism), indicating that the hormones induce a binucleating (polyploidizing) type of growth similar to that normally observed in the liver of growing rats. The highest proliferative activity (labeling index) in the hepatocyte cultures was found among the diploid cells, independent of the degree of mitogenic stimulation. The labeling index was inversely correlated with ploidy, suggesting that the ability of hepatocytes to proliferate decreases with increasing polyploidization.     

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.     

The induction of hepatocyte polyploidization in rats of various ages by ionizing irradiation with different LETs

A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of nonproliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation.     

An assessment of the relative contribution of the processes of cellular proliferation, polyploidization and hypertrophy to the increase in liver mass at various stages in the postnatal development of rats

Changes in isolated hepatocyte dry mass, its ploidy and liver mass at different stages of the rat postnatal ontogeny were investigated. The determination of these processes and special calculation made it possible to estimate quantitatively a relative contribution of cell proliferation, polyploidization and hypertrophy, not associated with DNA synthesis to the increase in the liver mass at different stages of the rat development. During the first week after the rat's birth, its liver growth is provided by 61 and 39% with hepatocyte proliferation and hypertrophy, respectively. Between the 14th and the 21st days of development, when considerable functional changes occur in the rat liver, the contributions of proliferative and polyploidization processes, and of cell hypertrophy into the liver mass increasing are roughly identical. Later on, the contribution of cell hypertrophy into the liver growth is noticeably reducing to reach within 1-2 months only 1%. On this developmental stage the liver mass increment by 2/3 is provided due to cell proliferation and by 1/3--to its polyploidization. As a whole, the accelerated growth of the rat liver from the birth to sex puberty is described as follows: the contribution of processes of proliferation and polyploidization, and of cell hypertrophy correspond to 28, 30 and 42%, respectively; afterwards, the liver growth being retarded. Within the period from 2 to 6 months, the liver mass increase is provided mainly (up to 76%) by cell proliferation, the shares of polyploidization and cell hypertrophy being 8 and 16%, respectively.     

Ploidy class-dependent variations during 24 h of glucose-6-phosphate and succinate dehydrogenase activity and single-stranded RNA content in isolated rat hepatocytes

The time-dependent variations over 24 h of glucose-6-phosphate dehydrogenase (G6PDH) activity, succinate dehydrogenase (SDH) activity and single-stranded RNA (ssRNA) content have been investigated by cytophotometric analysis of cytochemically stained isolated hepatocytes of different ploidy classes from adult male rats. A marked variation of 48 % over the day in G6PDH activity of the mononuclear diploid cells was revealed, but no significant variation in the binuclear tetraploid cells. The cells of the inbetween ploidy classes showed an amplitude of variation of 38 % (binuclear diploid cells) and 24% (mononuclear tetraploid cells), respectively. All cells showed a maximum activity of the enzyme at the middle of the day and a minimum during the night. The relative enzyme activity per mononuclear diploid cell was significantly higher than the relative activity in the other cells, especially at its maximum. The variation of the SDH activity in hepatocytes isolated from the same rats was similar in all cells, irrespective of their ploidy class. The activity was highest at the end of the activity phase of the animals. The SDH activity per cell was directly proportional to the quantity of genome copies. The ssRNA content of the hepatocytes showed a time-dependent variation with a maximum during the resting phase of the animals and a minimum during their activity phase. The variation was larger in the mononuclear diploid cells than in the cells of other ploidy classes and the ssRNA content was also significantly higher in these cells than in the hepatocytes of other ploidy classes when calculated on the basis of genome copies. It is concluded that the large amplitude of variation over the day and the high relative amount of G6PDH activity and ssRNA content in mononuclear diploid cells is related to the function of these cells as stem cells of the liver parenchyma.     

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.     

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.     

Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers. III. Cytological changes

Treatment of rats with cyproterone acetate (CPA) induces liver growth. Intact hepatocytes and cell nuclei were isolated from enlarged livers and their volumes or diameters were determined by electronic and microscopic methods. No changes in mean hepatocyte volume or ploidy were observed. However, there was a marked fall in the frequency of binuclear hepatocytes (from 43% to 7%) and a concomitant increase of nuclear ploidy. This effect probably resulted from CPA-induced replication of binuclear hepatocytes. The total number of hepatocytes replicating in response to CPA was estimated on the basis of these data and was found to be up to 75% of all parenchymal cells. Similar cytological changes were observed in the liver after treatment with pregnenolone-16 alpha-carbonitrile (PCN) and, to a lesser extent, with alpha-hexachlorocyclohexane (alpha-HCH). In contrast, physiological liver growth in adolescent rats was characterized by only small changes in binuclearity and nuclear ploidy, and by increases of cellular ploidy. Thus, ploidy analyses may be a useful tool to characterize the type of growth stimulation. Following discontinuation of treatment the cytological changes induced by CPA or alpha-HCH were not reversible in a matter of 3 weeks.     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.