Morphology and function of cultured hepatocytes isolated from rats with experimental toxic hepatitis

The goal of the study was to examine the morphology and function of primary hepatocytes isolated from rats with toxic hepatitis induced by a combination of CCl₄ and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell, and cell-matrix contacts. In this investigation, the morphology of mitochondria and their localization in hepatocytes was assayed with Rhodamine 123 staining. Glycogen and DNA contents in cultured hepatocytes were determined by fluorescent cytometry. It was found that the ploidy of hepatocytes isolated from normal and injured livers were different. Cells were maintained in culture for 5 days and no changes in ploidy distribution were observed. The glycogen content was 50% higher in the experimental group than the control one; it was decreased in control and cirrhotic hepatocytes treated with collagenase. Intact hepatocytes accumulated glycogen within 3 days; the glycogen level remained low in pathologic hepatocytes.     

Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.     

The cellular biology of megakaryocytes

Megakaryocytes show a pattern of cellular proliferation and maturation which is unique in mammalian biology. Cells mature to the point of cytoplasmic fragmentation in three major ploidy classes, 8n, 16n, and 32n and the three are fed from a precursor committed stem cell. Two-thirds of the cells belong in the 16n class, and approximately one-sixth in the 8n and 32n classes. The cytoplasm of cells in each ploidy class has a characteristic concentration of granules and demarcation membrane system which appears to be translated into the characteristic features of the platelet progeny from each class. These differ from normal young platelets. Megakaryocytes release fragments of cytoplasm into marrow sinusoids and these differ from platelets in that they do not have the peripheral microtubular bundle or sub-marginal dense tubular system. Transition from fragment to circulating platelet presumably takes place elsewhere in the circulation. With stimulation of platelet production, "stress" platelets are produced, from megakaryocytes which show changes with respect to content of polyribosomes, glycogen and membrane.     

Ploidy and nuclear area as a predictive factor of histologic grade in primary breast cancer

OBJECTIVE: To compare ploidy and nuclear area with histologic grade in breast cancer using cytologic samples. STUDY DESIGN: Fine needle aspirates from 85 patients with primary breast cancer were analyzed to identify ploidy and nuclear area. The Feulgen technique was used to stain the material. We used the SAMBA 4000 image analysis system (Grenoble, France) for analyzing ploidy and nuclear area. Each patient underwent a biopsy, and the histologic grade was analyzed. RESULTS: A significant association was found between ploidy and nuclear area, between histologic grade and nuclear area, and between ploidy and histologic grade. As ploidy became aneuploid and polyploid and nuclear area became larger, histologic grade became higher. CONCLUSION: A reliable and rapid evaluation of variables for breast cancer can be achieved using cytologic preparations by measuring ploidy and nuclear area of malignant cells with an image analysis system. Ploidy and nuclear area have a significant association with histologic grade.     

Some factors controlling step-wise variation of organelle number in Euglena gracilis

Both nuclear and plastid ploidy double during the first 2–3 cell generations during the phosphate-induced shift from half ploidy to full ploidy in mixotrophic Euglena. Growth conditions can affect nuclear and plastid ploidy differently; plastid ploidy is more labile than nuclear ploidy during heterotrophic growth of both wild type and mutant cells, while nuclear ploidy is more labile during photoautotrophic growth.     

Mechanism of ploidy shift from diploidy to tetraploidy in MSPC-1 mouse myeloma

In order to elucidate the cytological mechanism of ploidy shift from diploidy to tetraploidy in MSPC-1 mouse myeloma, the process of cell division was observed in living cells under phase contrast microscope. It was suggested that loss of cytokinesis and subsequent formation of binucleate cells are the major causes of such a ploidy shift. Elevated frequencies of binucleate cells during the transition phase of ploidy shift from diploidy to tetraploidy also supported the above notion. The possibility of cell fusion as a cause of the ploidy change could be eliminated by analyses of marker chromosomes and incorporation pattern of [³H]thymidine into binuclei.     

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.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.     

Variability of the cardiomyocyte ploidy in normal human hearts.

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19-39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2-3.9 c to 6.6-7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgen-stained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1 +/- 0.3 c, in the middle layer 5.5 +/- 0.3 c and in the inner layer 4.8 +/- 0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61 +/- 3%, 63 +/- 4% and 54 +/- 5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

中华蟾蜍糖原含量的季节变化

于2005年11月至2006年10月,用硫酸-蒽酮比色法和比重法测定了中华蟾蜍各月份的肝糖原和肌糖原含量及肝比重。结果显示:冬眠期间(11月至次年的2月),糖原含量逐月下降;2月份时出现临时回升,然后继续下降;4月份时肝糖原含量最低;5月份起,肝糖原含量逐渐上升;5月份时肌糖原含量为最低;6月份起,肌糖原含量逐渐上升。虽然在7-8月间出现过下降,两种糖原的含量在10月份时达到一年中的最高值。这些结果表明,蟾蜍糖原含量在一年中呈现显著的季节性波动。越冬前所储备糖原的一部分可能用于越冬期间维持高水平的血糖,一部分用于基础代谢。2月份时糖原含量的临时上升,可能是血液中作为防冻保护剂的葡萄糖运回肝脏和肌肉中再合成糖原的结果。7-8月间糖原含量降低可能与蟾蜍夏蛰有关。雌性5月至10月期间的肝糖原总体水平显著低于雄性,可能与依赖可得到葡萄糖的卵母细胞中的糖原合成有关。糖原含量的季节变化与蟾蜍的生活状态(越冬、繁殖等)有关,并与血糖含量有联动关系。     

Variability of the cardiomyocyte ploidy in normal human hearts

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19–39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2–3.9 c to 6.6–7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgenstained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1±0.3 c, in the middle layer 5.5±0.3 c and in the inner layer 4.8±0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61±3%, 63±4% and 54±5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Characterization of diverse ploidy in the arctic‐alpine Arenaria ciliata species complex (Caryophyllaceae) using shoot meristem staining and flow cytometry analysis of archived frozen tissue

Ploidy levels were analyzed in 21 European populations of the Arenaria ciliata complex using baseline chromosome counts derived from Feulgen staining of HCl‐treated shoot meristems and calibrated flow‐cytometry analysis of fresh and archival frozen tissue. Calibration with two to three control samples of different ploidy facilitated rapid identification of ploidy states in unknown samples. Observed ploidy levels varied from 2N = 40–200, with the majority of populations showing 2N = 40–80. High‐altitude populations collectively showed the full range of ploidy states, but at low elevations only lower ploidy levels were observed. Populations with the highest observed ploidy contained the greatest observed phylogenetic diversity in the western and eastern Alps. Multiple polyploidization events are inferred in the continental European metapopulation, with lower, more stable ploidy characteristic of the west and north. The method deployed provides an effective approach to ploidy analysis for archival desiccated/frozen tissue samples from biogeographic collections.     

Ploidy distribution in the explant tissue and the calluses induced during the initial stage of internode segment culture of Asparagus officinalis L.

Summary To pursue the causal factors of ploidy variation in tissue culture of asparagus (Asparagus officinalis L.), ploidy status of stem explants and the calluses induced from these explants were examined using flow cytometry (FCM). The frequency of higher ploidy (>8C) cells in the stem explants increased with increasing distance from the shoot apex. Calluses derived from sections far from the apex also showed higher ploidy distributions, and also higher ploidy (16C) in general than was observed in the explant tissue. Detailed studies using FCM and charge-coupled device (CCD)-equipped fluorescence microscopy showed that 16C cells appeared in the explant tissues within 1 wk of culture, possibly induced by the plant growth regulators (PGRs), especially 1-naphthaleneacetic acid (NAA), in the culture medium.     

Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary culture

Glycogen synthesis was examined in primary cultures of adult rat hepatocytes that had been isolated from rats following a 24-h fast. Glycogen synthesis was dependent on the concentration of glucose in the culture medium and also required the presence of insulin. The addition of dexamethasone to the culture medium also increased the amount of glycogen synthesis. When the culture medium was supplemented with [U-14C,3-3H]glucose, it was found that approximately 60% of the glucose incorporated into glycogen was not derived from the pool of labeled glucose. In addition, the relative ratio of 3H/14C in the newly synthesized glycogen was approximately 50% of the ratio of the two isotopes in glucose in the culture medium, indicating that the glucose had undergone metabolism prior to its incorporation into glycogen. However, when hepatocytes were isolated from rats that had been fed ad libitum and the synthesis of glycogen from [U-14C,3-3H]glucose was followed, the relative ratio of the two isotopes in glycogen was similar to that measured for glucose in the culture medium, indicating that the glucose was directly incorporated into glycogen without any apparent metabolism. These results indicate that the synthesis of glycogen from glucose may, at least in part, follow an indirect pathway whereby glucose is metabolized prior to incorporation of the carbon into glycogen, but that the pathway followed for the synthesis of glycogen is dependent on the prior metabolic state of the animal.     

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.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

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

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

Ploidy level of the invasive weed Rubus alceifolius (Rosaceae) in its native range and in areas of introduction

 A change in ploidy level could increase invasiveness of introduced plants in insular plant communities. To examine this question for R. alceifolius, we compared its ploidy level in its Asian native range and in the Indian Ocean islands where it has been introduced. We first counted chromosomes on root tips from a Vietnamese individual, which proved to be tetraploid (2n=4x=28). The nuclear DNA content of other individuals from the native range and areas of introduction was estimated using the flow cytometry method. The Vietnamese individual on which chromosomes were counted was added to the sample, to enable deduction of the ploidy level of all individuals from their nuclear DNA content. All individuals were found to be tetraploid, except 10 individuals from a single clone collected in a Vietnamese population, estimated to be triploid, and morphologically different of other individuals of this study. We showed that while polyploidy of the source population may have predisposed this plant to become a successful invader, its introduction into Indian Ocean islands was not associated with any change in ploidy level. Received January 23, 2001 Accepted May 22, 2001     

Ploidy effects on anatomy and gas exchange of tall fescue leaves

A growth chamber study was designed to interpret differences in CO₂ exchange rate (CER) and leaf diffusive resistance among 4X, 6X, 8X, and 10X ploidy levels of tall fescue (Festuca arundinacea, Schreb). Mesophyll cell size, stomatal density, number of major and minor veins, and bundle cap size of leaf blades were evaluated. Diffusive resistance tended to decrease and CER to increase with increasing ploidy level. Mean stomatal density decreased from 43.6 per square millimeter to 30.6 per square millimeter as ploidy level increased from 4X to 8X. The 10X ploidy level exhibited the highest stomatal density, 47.2 per square millimeter. Major veins decreased from a mean of 14.2 to 10.2, and minor veins increased from a mean of 4.2 to 6.6, per leaf blade as ploidy increased from 4X to 10X. Total number of veins decreased significantly from a mean of 18.4 to 15.7 as ploidy increased from 4X to 8X.     

Numerical taxonomy applied to a study of some ploidy levels within the Ornithogalum umbellatum complex (Hyacinthaceae) in France

A macromorphological study of certain ploidy levels within the Ornithogalum umbellatum complex was carried out in France. The data matrix was treated by multivariate analyses. Certain ploidy levels show clear morphological differenciation in relation to taxon provenance. In the Loire Valley, there is a very strong correlation between ploidy level of individual plants and a specific morph. The results are discussed from a systematic and evolutionary point of view within the framework of polyploid complexes to which O. umbellatum belongs.