Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.     

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.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.     

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 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)     

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

Synopsis 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 considerably with increasing nuclear ploidy.     

Liver cell polyploidization: a pivotal role for binuclear hepatocytes

Polyploidy is a general physiological process indicative of terminal differentiation. During liver growth, this process generates the appearance of tetraploid (4n) and octoploid (8n) hepatocytes with one or two nuclei. The onset of polyploidy in the liver has been recognized for quite some time; however, the cellular mechanisms that govern it remain unknown. In this report, we observed the sequential appearance during liver growth of binuclear diploid (2 x 2n) and mononuclear 4n hepatocytes from a diploid hepatocyte population. To identify the cell cycle modifications involved in hepatocyte polyploidization, mitosis was then monitored in primary cultures of rat hepatocytes. Twenty percent of mononuclear 2n hepatocytes failed to undergo cytokinesis with no observable contractile movement of the ring. This process led to the formation of binuclear 2 x 2n hepatocytes. This tetraploid condition following cleavage failure did not activate the p53-dependent checkpoint in G1. In fact, binuclear hepatocytes were able to proceed through S phase, and the formation of a bipolar spindle during mitosis constituted the key step leading to the genesis of two mononuclear 4n hepatocytes. Finally, we studied the duplication and clustering of centrosomes in the binuclear hepatocyte. These cells exhibited two centrosomes in G1 that were duplicated during S phase and then clustered by pairs at opposite poles of the cell during metaphase. This event led only to mononuclear 4n progeny and maintained the tetraploidy status of hepatocytes.     

Development of tetraploidy in V79 spheroids

Summary Chinese hamster V-79-171 cells, when placed in suspension culture, spontaneously form multicell spheroids. As the spheroids enlarge the fraction of polyploid (predominantly tetraploid) cells increases and can approach 100% in very large spheroids. Spheroid size, rather than age, seems to be a major determinant for increased ploidy. When cell separation techniques were used to select enriched populations of diploid and tetraploid cells, the growth rate and plating efficiency of the diploid cells was always marginally higher, and they gradually became predominant in mixed monolayer cultures. Cloned tetraploid cells, however, generally remained quite stable, and no consistent ploidy dependent changes in radiosensitivity were observed relative to normal, diploid cell lines. This research was supported by grants CA 28793 and CA 23511 awarded by the National Cancer Institute, Bethesda, MD.     

A cytophotometric and karyometric study of the cardiac muscle cells of young rats exposed to intermittent high altitude hypoxia

Thirty-day-old rats were exposed to intermittent high altitude in barochamber (7000 m, 4 h per day, 4 exposures) and number of mono- and binucleated myocytes as well as DNA content per nucleus were determined in the right ventricular myocardium. The data indicate enhancement of mitotic division of mono- and binuclear diploid cells in hypoxic animals. Simultaneously, an increase in mononuclear and binuclear tetraploid cells occurred; the latter is probably due to DNA synthesis not followed by mitosis of some binuclear diploid cells.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Summary Intoxication of rats with CCl₄ (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS⁺) hepatocytes were reestablished. However, while the GS⁺ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS⁺ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS⁺ and GS^– hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS⁺ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS^– hepatocytes. Immediately after liver damage by CCl₄ and during regeneration small but significant changes in the nuclear pattern were noted for GS^– hepatocytes. However, the first GS⁺ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS^– hepatocytes. These results indicate that new GS⁺ hepatocytes may be derived from formerly GS^– cells which are induced to express GS if they have reached the border of the central veins.     

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.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

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.     

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.     

Changes in pancreatic acinar cell nuclear number and DNA content during aging in the rat

Pancreatic acinar cells from rats 5 to 658 days (94 weeks) of age were isolated by enzymatic dissociation and stained with the DNA specific fluorochrome Hoechst 33258. The nuclear DNA content and the incidence of binucleation were estimated in these cells. Total pancreatic weight, RNA, protein and DNA, and the incorporation of 3H-thymidine into pancreatic acinar cell DNA were also estimated in similar animals as measures of pancreatic growth. From 5 to 17 days after birth, 95% of the cells were mononucleate diploid and 5% were binucleate diploid; but during the period of rapid pancreatic growth over the following 39 days, acinar cells became increasingly binucleate. By 56 days after birth, 64% of cells were binucleate with a diploid DNA content per nucleus; and the incidence of binucleation then remained constant. At 28 days of age, 4% of mononucleate cells were tetraploid, increasing to 6% at 658 days of age. At this time 3% of binucleate cells contained dual tetraploid nuclei. There is thus a rapid development towards diploid binucleate acinar cells in the growing, postnatal pancreas; and in the adult pancreas a small proportion of these cells develop tetraploid nuclei.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.