Journal of Histochemistry and Cytochemistry, Vol. 51, 319-330, March 2003, Copyright © 2003, The Histochemical Society, Inc.


ARTICLE

Quantitative Fluorescence Imaging Approach for the Study of Polyploidization in Hepatocytes

Eugenia Lamas1,a, Danielle Chassoux1,b, Jean-François Decauxc, Christian Brechota, and Pascale Debeyb
a Liver Cancer and Molecular Virology, Institut National de la Santé et de la Recherche Médicale, Unité 370, Paris, France
b Nuclear Dynamics and Development, Institut National de la Recherche Agronomique 806/EA 2703, Muséum National d'Histoire Naturelle, Paris, France
c Centre National de la Recherche Scientifique, UPR 1524, Meudon–Bellevue, France

Correspondence to: Danielle Chassoux, Institut National de la Recherche Agronomique 806/EA 2703, IFR 63, Muséum National d'Histoire Naturelle, IBPC, 13 rue Pierre et Marie Curie, 75005 Paris, France. E-mail: chassoux@ibpc.fr


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Materials and Methods
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Literature Cited

We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms. (J Histochem Cytochem 51:319–330, 2003)

Key Words: fluorescence microscopy, image analysis, computer assist, hepatocytes, polyploidization, DNA quantitation, Hoechst 33342, BrdU, double labeling


  Introduction
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Introduction
Materials and Methods
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Discussion
Literature Cited

IMAGE ANALYSIS of cells in fluorescence to extract quantitative data from biological observations was proposed over 10 years ago. Several groups have described conditions for quantification of nuclear DNA (Galbraith et al. 1991 ; Poulin et al. 1994 ; Wang et al. 1995 ). However, few studies have appeared in that field. CCD cameras have improved regarding sensitivity, speed, and spatial resolution and, together with faster computers and better software, they render cell biology studies more amenable to image analysis. In addition, fluorescence offers the unique advantage of multilabeling under nondestructive conditions. We consider here the case of polyploidization in normal hepatocytes. A feature of hepatocyte polyploidization is the occurrence of binucleated tetraploid and octoploid cells together with mononucleated cells at each ploidy level. Hepatocyte polyploidization is associated with the appearance of markers of terminal differentiation and senescence, accompanied by a progressive decline in cell proliferation and a higher sensitivity to apoptosis (Sigal et al. 1999 ). Binucleated cells are from the beginning preferentially located in the centrolobular area (LeBouton 1976 ), whereas diploid cells are predominantly located in the periportal area and retain their proliferative capacity. The precise mechanisms involved in polyploidization are still not clear. Multiple labeling coupled with image analysis appears to be a promising method to gain new insights in this field.

Hepatocyte ploidy has been investigated in the past essentially through karyometry (Nadal and Zajdela 1966 ) and cytophotometry (An et al. 1997 ). More recently, several groups have used flow cytometry (FACS) (Gerlyng et al. 1993 ; Sigal et al. 1995 , Sigal et al. 1999 ). However, FACS cannot resolve nuclearity and a second step is required, in which a fraction of a gated population is screened under the microscope (Hasmall and Roberts 1997 ). Few reports have taken advantage of fluorescence imaging to directly assess nuclearity and measure DNA content and, in combination with BrdU staining, to analyze growth and binucleation rate on isolated hepatocytes in culture (Mossin et al. 1994 ).

We set out to see if DNA quantitation could be precisely performed by fluorescence imaging on liver cell preparations so that quantitative analyses could be performed at the single-cell level in situ. For this we require well-separated whole flattened cells. These experiments are intended to serve in multiparametric analyses to study gene expression and protein localization (Linares-Cruz et al. 1995 ) with respect to ploidy. For these reasons we chose to use crosslinking fixatives that best preserve the native localization of cell constituents (Visser et al. 1998 ; Tumbar et al. 1999 ) and Hoechst 33342 to stain DNA, because this compound, which binds stoichiometrically to DNA when crosslinking fixatives are used, does not require DNA denaturation or RNase treatment. We hypothesized that enzymatically dispersed liver cells allowed to adhere and spread before fixation would be suitable material, for the following reasons. First, we found fluorescence imaging to be a reliable method for DNA quantitation when performed on fibroblasts in culture. Measurements were sufficiently precise that an increase in DNA content was observed during the different stages of S-phase, as recognized by the typical labeling patterns after BrdU incorporation (Chassoux et al. 1999 ). Second, liver cells isolated by enzymatic treatment constitute a widely used cell preparation method in the field of hepatology and there is no evidence to indicate that this might preferentially isolate one of the ploidy classes present in the normal liver, as assessed by flow cytometry or image analysis. Moreover, when liver cells are in suspension, tissue heterogeneity is abolished, so that a sample is representative of the whole population.

The aim of this study was to demonstrate that nuclear DNA content could be precisely measured under the selected experimental conditions and that the cells in the various nuclearity and ploidy classes could be adequately identified within a total number of cells compatible with image analyses. In addition, we show that primary short-term culture of liver cells offers access to information on the speed of the cell cycle according to cell class.


  Materials and Methods
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Materials and Methods
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Preparations of Hepatocytes
Rats were purchased from IFFA-CREDO (Lyon, France). They were all fed the same standard diet. Hepatocytes were isolated from male Wistar rats by cannulating the portal vein and perfusing the liver with Liberase-calcium (Liberase Purified Enzyme Blend; Boehringer–Mannheim, Mannheim, Germany). The ages of animals ranged from birth up to 12 weeks. After isolation, cells were collected in L-15 medium enriched with 1 mg/ml bovine serum albumin (BSA) and left to sediment for 20 min at room temperature (RT). After three washes in the same medium, liver cells were seeded at 1.106 cells/cm2 on pretreated glass coverslips in Williams medium E, supplemented with 10% fetal bovine serum (FBS) and 1 mg/ml BSA at 37C in a 5% CO2 atmosphere (McIntyre et al. 1999 ). Preparations with less than 5% dead cells were used. No selective enrichment for hepatocytes was performed.

After cell attachment (4 hr in medium with serum, referred to as Step 1 in the text), the medium was removed and replaced by fresh, serum-free Williams medium E containing 0.5 mg/ml BSA, 5 mg/ml bovine insulin, and 7 x 10-7 M hydrocortisone, hemisuccinate, and the cells were cultured for a further 12 hr (Step 2). All culture media used contained penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (250 ng/ml). These cell preparation conditions were designed to obtain flattened cells suitable for image analysis.

In some experiments, cells were seeded on collagen and kept in serum-free medium for 24 hr before being submitted to mitogenic stimulation by culturing them in medium supplemented with 50 ng/ml epidermal growth Factor (EGF) and 20 mM sodium pyruvate (McIntyre et al. 1999 ). Appositions were done by approaching Superfrost slides (CML; Nemours, France) to a fragment of liver tissue. Preparations were air-dried at RT for 24 hr before fixation.

Cell Fixation
At the end of the culture period, the cells were washed three times in PBS and fixed in 4% PFA in PBS (PFA) for 10 min at RT. After extensive washing, cells were stored in 1% BSA in PBS or in 70% ethanol at 4C.

Appositions were fixed in PFA for 15 min at RT and washed in PBS before staining. For flow cytometry (FCM), cells suspensions (before seeding on coverslips) were fixed in 70% ethanol and kept at 4C.

DNA Staining
Cells were incubated in 2 µg/ml Hoechst 33342 (H42) (Riedel de Haen; Seele, Germany) in PBS for 30 min at RT before being washed and mounted in Permafluor (Immunotech; Marseille, France). The compound H42 has been shown to bind stoichiometrically to DNA when crosslinking fixatives are used (Santisteban et al. 1992 ).

For FCM, cells were treated with RNase and stained with 10 µg/ml propidium iodide (PI). FCM was performed on a FACStar-plus (Becton–Dickinson; Mountain View, CA).

BrdU Incorporation and Detection
Rats were injected intraperitoneally with bromodeoxyuridine (BrdU) (Sigma; St. Louis, MO) (30 mg/kg body weight) 1 hr before sacrifice. After hepatocyte isolation, cells were cultured either with (10 µM) or without BrdU. Both preparations were fixed at the same time, at the end of the culture period.

Double staining for BrdU and total DNA was performed in conditions that do not modify DNA content measurements (Dolbeare et al. 1983 ). Cells were treated with 2 N HCl for 20 min at RT and carefully washed in PBS before immunostaining using a mouse monoclonal anti-BrdU antibody (Caltag Laboratories, Burlingame, CA; dilution 1:100) and an FITC-conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Chester, PA; dilution 1:200). The cells were then stained with H42.

DNA Content Measurements and Detection of BrdU Incorporation in the Same Cells Using Fluorescence Imaging
The cell preparations were examined under a Zeiss (Axiovert 35) (Carl Zeiss; Gottingen, Germany) inverted microscope equipped for epi-illumination (50-W mercury lamp). Zeiss Plan Neofluar objectives x20 (NA 0.5) and x40 (NA 0.75) were chosen, enabling the collection of light from the entire thickness of the nucleus, since these conditions are essential for adequate DNA content determinations (Arndt-Jovin and Jovin 1989 ). The following Zeiss filter blocks were used: for H42, excitation bandpass 365/11 nm, dichroic mirror 395 nm, emission bandpass 450–490 nm; for FITC, excitation bandpass 450–490 nm, dichroic mirror 510 nm, emission longpass 520 nm.

Images were captured using a cooled CCD camera (Photometrics, Tucson, AZ; KAF 1400-G2, class 2), on 4056 gray levels as described (Chassoux et al. 1999 ). Briefly, images were captured sequentially, Hoechst images always taken first. Exposure time was 0.5 or 1 sec for H42 and 5 sec for FITC. Automatic quantitative image analysis was performed in 12 bits using IPLab Spectrum version 3.1 software (Scanalytics; Fairfax, VA) running on an 8500 Power Macintosh computer. Correction for illumination inhomogeneity was performed with the "Flat field" function, using the image given by a fluorescent crystal (Carl Zeiss) as "Uniform window" and a black image as "Dark." Segmentation was done by one-step thresholding after correction of the image. In conditions under which most cells are viable at fixation, Hoechst stain does not give any cytoplasmic signal. Hepatocyte autofluorescence is detected at a wavelength different from that of Hoechst (Benson et al. 1979 ), thus not affecting the positioning of the threshold for the segmentation of nuclei stained with Hoechst. Nuclei were assigned to a mono- or a binucleated cell by comparing fluorescent and brightfield images. Occasional overlapping nuclei or debris were eliminated interactively by inspection on the screen, using zoom and contrast enhancement where necessary.

Parameters such as integrated fluorescence (IntF) or area of selected objects were stored in computer files for analysis using Kaleidagraph. The IPLab software also provides a color code giving, from dark blue to red, the distribution of values from the lowest to the highest value. A total of 300–400 cells were studied per timepoint, observed on 8–12 separate fields. This number is well in the range of other studies based on imaging (Murata et al. 2001 ). To ensure that the same cells were not counted twice, slides were read in a systematic manner, moving from top right to left of the slide and then on successive descending lines.

For BrdU labeling, cells were inspected on the computer screen and scored as BrdU-positive by the operator when bright-green fluorescent dots were seen over the nuclear area, as identified by Hoechst stain. Cells scored as negative for BrdU displayed evenly dark nuclei, even after artificially increasing the contrast of the image. BrdU labeling patterns were recognized and classified according to the established nomenclature of the various stages of the S-phase, each recognized by typical distributions of the replication sites: early S-phase, many granules of small size distributed all over the nucleus, excluding nucleoli, not reaching the border ("early"); mid-S-phase, clustering of fluorescent signals around nucleoli and at the periphery of the nucleus ("mid"); late S-phase, fewer signals of larger size distributed in the interior of the nucleoplasm ("late") corresponding, in rodents, to heterochromatin-rich regions (Nakayasu and Berezney 1989 ; O'Keefe et al. 1992 ). Morover, it is known that the patterns acquired on pulse labeling at a given stage of the replication process are maintained during progression through S-phase and the following cycles (Ma et al. 1998 ; Jackson and Pombo 1998 ).

Statistical Analyses
These were performed using the Chi-squared test or the nonparametric test of Mann–Whitney (level of significance p< 0.05).


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Ploidy Measurements by Fluorescence Imaging
Liver cell preparations from adult rats were fixed and stained for examination by fluorescence imaging. The analytical procedure is illustrated in Fig 1. Hoechst-stained nuclei appeared brightly fluorescent and could be assigned to a given cell by comparison with the corresponding brightfield image (Fig 1A and Fig 1B). Automatic quantitative measurement of Hoechst fluorescence intensity was performed after correction of the image and, depending on the value of this parameter, each nucleus was assigned a false color. As shown in Fig 1C, the color scale indicates that nuclei have integrated fluorescence (IntF) values centred around three values only. The smallest nuclei in mononucleated cells appear blue. Nuclei in binucleated cells, when considered as one object (by linking them artificially), appear to have the same color as larger nuclei in mononucleated cells (green) (Fig 1C and Fig 1D, right panel). When considered individually, each nucleus in a binucleated cell appears the same color as the smallest nuclei in mononucleated cells (blue) (Fig 1D, middle panel). The nucleus of one large mononucleated cell appears red. A typical histogram of data obtained is shown in Fig 2A. The first peak shows a coefficient of variation (CV = standard deviation/mean) of 5.8%. The second peak is at a position twice the value of the first peak. Considering mononucleated cells, DNA index (mean IntF of second peak/mean IntF of first peak) is 2.04. When the two nuclei in binucleated cells are analyzed as one object (hatched columns), there is a complete overlap of their IntF values with those of the mononucleated cells in the second peak (open columns). In contrast, when binucleated cells' nuclei are analyzed separately, their IntF values attain those of the mononucleated cells in the first peak (ratio of the means was 1.04 for 28 elements in this example). From these data we conclude that the first peak represents 2C cells nuclei and the second peak 4C cells nuclei [mononucleated 4C (4Cm) and binucleated 2x2C]. The CV (indicative of the precision of the measures) value as well as the DNA index (indicative of the accuracy) indicate the high probability of the separation of the first two peaks. In this field, a nucleus was found at twice the value of the second peak. This suggests that this cell belongs to a rare population of mononucleated 8C cells. Histogram representations of other DNA content measurements showed, without any exception, similar distributions of IntF values for each ploidy level, without intermediate values. The CV of 2C values ranged from 4% to 8% and DNA indexes ranged from 1.95 to 2.05 for the 8–12 fields examined in each of four rat preparations.



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Figure 1. Illustration of the analytical procedure in a preparation of liver cells from a young adult rat. Fluorescent nuclei stained with Hoechst (A). Three overlapping nuclei (arrowhead) were excluded from the analysis. Close nuclei (arrow) belonging to two different cells as seen in the brightfield image (B) were analyzed as separate objects. Bar = 10 µm. (C) Color code for integrated fluorescence. The smallest nuclei in mononucleated cells appear blue; nuclei in binucleated cells (analyzed as one object) and larger nuclei in mononucleated cells appear green. One nucleus, largest in size, appears red. (D) Superimposed fluorescence and brightfield images (left): nuclei in binucleated cells, when analyzed first as separate objects (middle), then appear the same color as nuclei in mononucleated cells (blue), indicating diploidy. When analyzed as one object (by joining them where necessary, right), nuclei appear green, indicating tetraploidy. x20 objective. Cell size is half that in A–C.



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Figure 2. (A) Histogram of DNA content distribution. Cells from an 8-week-old rat liver cell preparation were allowed to adhere and then spread on glass in the absence of serum. There is a discontinuous distribution in three peaks, representing 2C, 4C, and 8C cells. The second peak comprises binucleated cells (hatched columns) and mononucleated cells (open columns). (B) Plot of nuclear area vs DNA content, showing a wider dispersion of nuclear area data irrespective of cell type. For diploid cells (n=28), CV area=17.4%, CV integrated fluorescence=5.8%. Binucleated cells (open circles), mononucleated tetraploid cells (diamond-shapes), mononucleated octoploid cell (circled diamond). Same added fields as in A. Nuclei in binucleated cells were analyzed as one object. Integrated fluorescence is in arbitrary units. x20 objective.

Nuclear area values showed a wider dispersion than fluorescence intensity values. This applied to diploid cells as well as to tetraploid mononucleated and binucleated cells (Fig 2B). It was probably due to the different relative spreading of the cells within a group of cells and did not affect fluorescence intensity measurements. The mean area did increase with ploidy. In the example, the 4C cells' mean nuclear area increase over the 2C cells' mean nuclear area was 1.75.

These findings, several ploidy levels, absence of S-phase, binucleated cells, and increase in nuclear size with DNA content, are known features of adult rodent hepatocytes. This indicates that we were able, in the cell preparation and imaging conditions used here, to analyze correctly various parameters in the cell population. Regarding quantitative measurements, the size of the CVs of DNA content measures was satisfactory.

We then proposed to measure the DNA content of cells in S-phase. Adult rat liver cell suspensions spread on glass and cultured in mitogenic medium are known to re-enter S-phase with a characteristic timing (Mossin et al. 1994 ; McIntyre et al. 1999 ). We made use of this system to further document the precision of the DNA content measurements of liver cell nuclei by fluorescence imaging. In addition to providing cells in S-phase, these preparations provide cells in conditions resembling as much as possible the conditions we used previously.

Cells were fixed for 23 or 28 hr after mitogenic stimulation. BrdU, added to the medium 45 min before fixation, was revealed by indirect immunofluorescence and cells stained with Hoechst. "Early" and "mid" labeling patterns were recognized at both time points examined, indicating that cells are not truly synchronous in this experimental condition. Data were pooled, building up an overall level of proliferation of 13%. The DNA contents of doubly stained nuclei were found in between the ploidy peaks defined by BrdU-negative nuclei. This is illustrated in Fig 3A and Fig 3B, in which two fields were added. Nuclei displaying an "early" labeling pattern (discrete small foci distributed all over the nucleoplasm, excluding nucleoli) had IntF values to the right of the mean of the 2C, 4C, or 8C peak on the histogram. An increase in DNA content up to 23% above the mean value of the previous peak was measured in BrdU-positive nuclei (n=31). "Mid" labeling pattern (clustering of foci around nucleoli and at the nucleus periphery) corresponded to an increase in DNA content of 30–87% (n=27) above 2C, 4C, or 8C mean value. These values also include rare nuclei with a "late" labeling pattern. A total of 563 cells were studied over 29 fields. In these experiments, the quantitative measurements were done at x40. The CV of 2C values of BrdU-negative nuclei ranged from 5% to 7% and DNA indexes ranged from 1.95 to 2.1. These results indicate that we can detect cells in S-phase by IntF measurements. BrdU-positive nuclei appeared as different colors from BrdU-negative nuclei on the color code for DNA content (not shown).



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Figure 3. Liver cell preparation of a 6-week-old rat, cultured in mitogenic medium for 23 or 28 hr and pulsed with BrdU for 45 min before fixation. (A) Histogram representation of DNA content in BrdU-negative (open columns) and BrdU-positive (filled columns) nuclei. (B) Scatter plots of DNA content vs nuclear size in BrdU-negative (open circles) and BrdU-positive (filled circles) nuclei, showing a progressive increase in size of nuclei in S-phase. Same added fields as in A. BrdU-positive nuclei have IntF values in between 2C, 4C, and 8C IntF values of BrdU-negative nuclei. One BrdU-positive nucleus has an IntF value higher than 8C. Nuclei in binucleated cells were analyzed as one object and IntF data pooled with those of 4Cm cells. CVs of BrdU-negative 2C and 4C nuclei are 5.4% (11 elements) and 3.7% (28 elements), DNA index 2.05. Integrated fluorescence is in arbitrary units. x40 objective.

Percentages of Cells in the Various Nuclearity and Ploidy Classes
Cells characterized in their ploidy and nuclearity could then be counted, collecting data from several microscopic fields to reach a number of total cells higher than 300.

Liver cell preparations from 6-day-old up to 12-week-old rats were analyzed. The proportions of cells in the nuclearity and ploidy classes are given in Table 1. Tetraploid cells were found as early as six days after birth at a frequency of 1%. They became the major population from 6 weeks onwards. Seventy percent tetraploid cells were found in 6-week-old rats by FCM, a similar percentage to that given by fluorescence imaging when adding up 2x2C and 4Cm populations. Binucleated 2x2C cells were also detected as early as 6 days of age, with a gradual increase in numbers up to 4 weeks and then a slow decline towards a value of around 20% at 3 months. When binucleated cells on appositions were counted, a lower percentage was found compared with fluorescence imaging. For example, at 21 days of age we found 6% 2x2C cells, whereas 4Cm cells numbers were similar to those found by fluorescence imaging. Octoploid cells arose as soon as 21 days after birth, with higher numbers for 2x4C than for 8C cells after 1 month of age.


 
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Table 1. Relative proportions of cells in the various ploidy and nuclearity classes with age (%): DNA content was measured by quantitative fluorescence imaging of nuclei of Hoechst-stained rat liver cells

The percentages were established from a total of 300–400 cells because we observed that no more significant information could be gained beyond 200 cells. This is indicated as follows. For each field, the total number of cells and the relative proportions in nuclearity and ploidy vary. As an indication of the inference of the sample size to the cell counts, we calculated the increase in precision provided by adding up fields. Data from a preparation of a rat just over 3 weeks old are presented as an example, studying nine fields (total number of cells 377). We calculated the cumulative percentages of 2C or 4C mononucleated cells, starting from any one of the nine fields (Fig 4). For frequent events (2C=60%) or less frequent events (4Cm=10%), the addition of five fields (mean number of cells n=196, range=187–249) gives information that is statistically no different (Chi-squared test) from the addition of eight fields (n=363, range=293–345).



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Figure 4. The cumulative percentages of 2C (A) and 4C (B) cells starting from any one of the nine fields (B–J) considered. Preparation from a rat over 3 weeks old, in which 2C cells were 62% and 4C mononucleated cells were 10%. The distribution of percentages is not significantly different when five fields are summed or when eight fields are summed (CV 5% vs 3% for 2C cells, 9% vs 6.5% for 4Cm cells, respectively). Chi-squared test.

The limit of sensitivity of the assay can be placed at 1 in 300 because a minimum of 300 cells is analyzed at each age point and rare events are undoubtedly detected (Fig 1; Table 1).

Distributions of DNA Content Values at Various Ages
The DNA content histograms for liver cell preparations at given ages were established (Fig 5). These are representative of all the preparations presented in Table 1. The size of the CVs in the young did not appear to be larger than that in the adults. In each case, a discontinuous DNA content distribution was observed, suggesting that there was no cell in S-phase. This was surprising in a situation of rapidly expanding liver mass. The possibility that sampling could be a problem here was considered and we decided to search for cells entering the cell cycle by detecting incorporation of BrdU.



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Figure 5. Analysis of the ploidy distribution of hepatocytes at given ages after birth. Histograms of DNA content distributions, showing an absence of S-phase and little dispersion of integrated fluorescence values. (A) Six-day-old and (B) 19-day-old: occurence of a tetraploid class. In older rats, the predominent class is tetraploid and 8C cells are found. (C) Four-week-old rat and (D) 6-week-old-rat. Integrated fluorescence x 105. x20 objective.

Evidence for DNA Synthesis
BrdU was injected before sacrifice and also, after hepatocyte isolation, BrdU was added to the culture medium and left throughout the experimental time. The percentages of positive cells scored at days 13, 19, 28, and 42 were respectively 32%, 20%, 14%, and 1%. These values appeared surprisingly high and we wondered if there was S-phase induction during the culture step. We next designed experiments to investigate the contribution of each culture step to the observed level of BrdU incorporation. Cells were split into four groups, receiving BrdU for either one only of the subsequent culture steps (Step 1 or Step 2) or both of them and a control group that did not see BrdU in culture. Detection of incorporated BrdU was done, for each group, at the end of the culture period. Only in the youngest rats did the number of positive cells increase over that found by in vivo incorporation (50% increase, two experiments). Therefore, those cells ready to enter S-phase were able to do so during Step 1, indicating that their viability was not affected by the preparation procedure. In the absence of serum (Step 2), no further DNA synthesis took place so that the level of BrdU incorporation in the "Step 1 + Step 2" group was the same as for the "Step 1" group. Thus, mononucleated diploid cells, representing the vast majority of cells at this age of the animals (13 days), actively proliferate and engage in S-phase asynchronously, even in vitro, but only when serum is present. Fig 6 shows a BrdU-positive mitotic figure (late telophase) in a 13-day-old rat liver cell preparation taken from the "Step 1 + Step 2" group. With the possibility of the onset of S-phase taking place in vitro at that age, the observation of labeled mitoses suggest that the duration of S and G2+M is of 12 hr minimum. Therefore, the completion of a cell cycle may take place in vitro, giving rise to two labeled daughter cells, so that the labeling due to BrdU incorporation took place during the S-phase of the preceding cycle.



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Figure 6. In vitro occurrence of mitoses. (A) Late telophase stage, positive for BrdU, is shown. Four interphase nuclei are present in this field, one of which is BrdU-positive. (A) Indirect immunofluorescence; (B) phase-contrast. Bar = 10 µm.

Comparisons of DNA Content and DNA Synthesis
The apparent discrepancy between BrdU positivity and the corresponding DNA content measurements in young rat liver cells (Fig 5A–5C) was addressed next in double labeling experiments in which total DNA content (stained with H42) was measured on the same cells in which incorporated BrdU was detected.

Pulse Labeling and BrdU Labeling Patterns. BrdU was given in vivo only (pulse) to get labeling patterns characteristic of the successive stages of S-phase. Fixation was done as usual at the end of the culture step. This situation is equivalent to a pulse chase experiment. BrdU-positive cells were found that displayed 2C, 4C, or 8C DNA content. This is indicated in Fig 7 by the color code in which blue is ascribed to 2C cells, green to 4C cells, and red to 8C cells. The various types of replication patterns were recognized in BrdU-positive nuclei. These were found in each of the nuclearity and ploidy classes, indicating that there was no selective loss of one of the classes nor loss of viability, during the observation period, of cells engaged in S-phase.



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Figure 7. BrdU patterns in the various ploidy and nuclearity classes. Preparation from a 28-day-old rat. BrdU was administered 1 hr before sacrifice and cells were fixed after 16 hr of culture. Color scale shows 2C as blue, 4C as green, and 8C as red. (A) Two diploid cells, identified by transmitted light (Phase) and on quantitative analysis (IntF), display a "mid" pattern of nuclear labeling (BrdU). A binucleated cell, negative for BrdU, is identified with H42 stain. Two out-of-focus BrdU-positive cells would not be considered, as DNA content measurement is then incorrect. (B) A mononucleated tetraploid cell positive for BrdU is seen displaying an "early" pattern of DNA labeling. Other cells in this field, BrdU-negative, are three binucleated tetraploid cells (same color as the mononucleated tetraploid cell) and one diploid cell, appearing as blue on quantitative analysis of Hoechst fluorescence (IntF). (C) A binucleated octoploid cell positive for BrdU is seen displaying an "early" pattern of labeling. For quantitative analysis, the two nuclei of the binucleated octoploid cell (identified by observation with transmitted light) have been joined to indicate, with the color code, the total DNA content in that cell (red) compared with the DNA content of BrdU-negative cells: a binucleated tetraploid (green) and a diploid (blue) cell. Bar = 10 µm.

Diploid Cells All three patterns were recognized, indicating asynchrony and cell cycle completion, regardless of the stage of diploid cell engagement in S-phase (labeled during S-phase, they were observed with a 2C DNA content). They were often seen as pairs of cells, whose nuclei exhibited similar labeling patterns (Fig 7A). Seventeen percent of the diploid cells were labeled at days 19 and 28, representing, respectively, 74% and 61% of all labeled cells.

Other Ploidy Classes Among the mononucleated 4C cells, half of them were labeled at day 19, representing 25% of all labeled cells. At day 28, 12% were labeled for BrdU, representing 12% of all labeled cells. Most of them displayed an "early" labeling pattern (Fig 7B), although other patterns were also seen. Labeled at the onset of S-phase (of a 2C nucleus), they were observed with a nuclear DNA content of 4C. This demonstrates that they progressed through S-phase during the experimental time [the labeling pattern acquired on pulse was retained during the next S-phase stages (Jackson and Pombo 1998 ; Ma et al. 1998 ) and that they must have arrested at this moment of their cycle because a DNA content of 4C was observed 17 hours later].

Binucleated 2x2C hepatocytes positive for BrdU displayed a late pattern of staining. These cells were labeled during the preceding S-phase (of a 2C cell) and were likely formed during the culture step. This was a rare event (6% of labeled 2x2C versus total 2x2C cells at 19 and 28 days), suggesting that the binucleation process is a slower process than the division of a diploid cell into two daughter diploid cells. Binucleated 2x4C hepatocytes displaying "early", "mid," or "late" replication patterns were observed (Fig 7C and Fig 8), suggesting that, as in the case of mononucleated tetraploid hepatocytes, the cycle was arrested before mitosis. Most of the 2x4C cells (83%) were BrdU positive at day 28. Therefore, the absence of S-phase in the DNA content measurements is now explained by the fact that cells completed DNA synthesis during the culture step, with some cells pursuing the cycle towards mitosis and G1, as confirmed by the following observations.



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Figure 8. Same preparation as in Fig 4. A binucleated octoploid cell positive for BrdU is seen, displaying a "mid" pattern of labeling, with clustering of sites labeled by BrdU at the periphery of the nucleus. H42 stain demonstrates nuclear domains of condensed chromatin, around which some of the sites labeled by BrdU are clustered. The two nuclei are far apart. Bar = 100 µm.

Continuous BrdU Labeling and Quenching of Hoechst. In the experiments where BrdU was added continuously in culture, we noted that, in 2C cells positive for BrdU, DNA content measurements decreased by 15–20% compared with 2C cells negative for BrdU present in the same field. This difference in IntF values was statistically significant (Mann–Whitney test). A shift to the left of the G1 peak, as shown in Fig 9, is typical of the classical phenomenon of quenching of Hoechst fluorescence by BrdU (indicative of a cell cycle completion after BrdU substitution during the entire preceding S-phase). A 20% quenching measured by FCM was reported with a 10 µM BrdU concentration (Kubbies and Rabinovitch 1983 ). We have observed, by in situ fluorescence imaging, a quenching of Hoechst by BrdU of a level similar to that of hepatocytes in NIH-3T3 fibroblasts in culture, and this will be reported elsewhere. The observation made here on hepatocytes further supports the interpretation of the double-labeling experiments mentioned above: BrdU positive diploid cells completed a cycle during the culture step, with a duration of S+G2+M below the experimental time (17 hr).



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Figure 9. Quenching of Hoechst by BrdU. Hepatocyte preparation from a 13-day-old rat, BrdU present throughout the experiment. A 4C cell, BrdU-negative, is measured at twice the mean IntF value of the 2C cells. The decrease in IntF values displayed by BrdU-positive cells (hatched column) vs BrdU-negative cells (black column) is statistically significant (Mann–Whitney test). x40 objective.

In these continuous labeling experiments, BrdU-labeled cells appeared brightly positive all over the nucleus, and labeling patterns could not be recognized any longer. This is another indication that the cells have continued in S-phase in vitro.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

We have shown here that, under conditions compatible with optical microscopic image analysis, liver cells in the various ploidy and nuclearity classes are indeed correctly identified. The proportions that we observed in adult rats are in agreement with results obtained by others with larger numbers of cells. The preparation conditions used in previous studies were designed for ploidy alone or, in some instances, for ploidy and S-phase. Here we favored whole-cell preservation, with a view to future studies on gene expression and protein localization.

Analyzing ploidy on appositions of mouse liver, Santisteban et al. 1992 had shown that, using Boehm–Sprenger fixation, Hoechst staining yielded the narrowest CV of all the DNA stains tested (6.68% for the 2C peak) and DNA index was 2.0. Using PI as DNA stain yielded higher CVs, so that S-phase cells cannot be identified by DNA content alone with this dye under the fixation conditions used. This is also apparent from other studies (Mossin et al. 1994 ) and from measurements performed on a laser scanning cytometer (LSC) (unpublished observations). This would be a drawback to using the LSC; moreover, the LSC cannot resolve nuclearity nor distinguish overlapping nuclei.

Analyzing appositions, we found that binucleated cells (not assessed in Santisteban et al. 1992 ) were underestimated compared with cells left to adhere and spread on coverslips. This is most likely related to the fact that the outer limits of the cytoplasm are difficult to see at low magnification under phase-contrast when cells are too tightly distributed. We obtained for tetraploid cells values similar to those reported before by fluorescence imaging using cytospin preparations of enzymatically dispersed hepatocytes and counting more cells. Mononucleated tetraploid cells become predominant over the binucleated cells at 6 weeks and later, whereas they are less numerous than the binucleated at 4 weeks. In 6- to 12-week-old rats, the reported range of tetraploid cells given by FCM is similar to our fluorescence imaging data (2x2C and 4Cm cells were considered as one population).

Ploidy classes for younger rats are little documented. Data obtained by karyometry on squashed tissue (Nadal and Zajdela 1966 ) or by counting cells in liver sections (LeBouton 1976 ) are in agreement with ours regarding binucleated hepatocytes. The occurrence of tetraploid cells, mono- and binucleated, in suckling rats appears to have been missed in FCM analyses (Sigal et al. 1995 ). The number of mononucleated tetraploid cells we found at day 20 was higher than that found by Nadal and Zajdela 1966 . This may be due to a more precise detection of this cell class by fluorescence imaging than by karyometry or possibly to an earlier requirement for tetraploidy, related to factors such as food or housing. We also observed an earlier shift towards mononucleated tetraploid cells than in their study (6 weeks vs 8 weeks).

The relationship between binucleated and mononucleated polypoid cells is not clear. The largest numbers of binucleated cells are found earlier than that of 4Cm. This observation is compatible with the scheme according to which, in binucleated cells, a concomitant DNA synthesis takes place in the two nuclei (BrdU-positive 2x4C cells were found at day 28), and then there is constitution of a common spindle and mitosis, giving two mononucleated tetraploid cells (Nadal and Zajdela 1966 ). However, the possibility that 4C cells arise concomitantly (by G2 arrest) cannot be excluded. It should be recalled that we found BrdU-negative 4Cm very early on and mononucleated 8C cells as early as 2x4C cells (in the absence of 2x8C), suggesting a possible pathway for polyploidization other than the binucleated cell. Yet another alternative hypothesis would be that a single molecular mechanism could lead, initially, to binucleated tetraploid (2x2C) cells and then to mononucleated tetraploid (4Cm) cells. The asynchrony demonstrated by BrdU in each of the ploidy and nuclearity classes has been, until now, little documented in young rats liver cells. This makes it difficult to deduce cell of filiation by just considering the evolution of the percentages of different cells with age.

An important point is that rare events (one cell in several hundreds) are unambiguously identified. This is permitted by the size of the CV and indicates that no information is lost due to the number of cells analyzed. It allows us to address situations such as physiological liver growth, presenting a low level of proliferation, with an assay keeping observations as close as possible to the in vivo situation. Pathological samples (fine needle aspirates, for instance) could also be analyzed by fluorescence imaging, in addition to (or in place of) FCM.

It should be stressed that static DNA content measures are not able, as such, to account for the events taking place in the young rat liver, because an active proliferation gives rise to G2 cells that cannot be discriminated, by DNA content alone, from mononucleated 4C polyploid hepatocytes. The short-term assay used in this study allowed rapidly cycling cells to proceed through S-phase to the G1 position of the next cycle, explaining why, in young rats, we found higher numbers of cells having incorporated BrdU compared with in vivo tritiated thymidine pulse experiments (Post et al. 1963 ). Therefore, cells found with a 4C nuclear DNA content at the time when preparations were fixed displayed a longer cell cycle period or a temporary cell cycle arrest, events building up during (BrdU-positive cells) or before (BrdU-negative cells) the assay. The BrdU patterns, that were made use of in these experiments to determine at which stage of S-phase the BrdU was incorporated could not be observed in longer labeling experiments. Longer labeling appears to be dispensable for looking at proliferation in liver cells. The discrepancy between DNA contents measurements and BrdU patterns was due to the prolongation of the cells in the living state in vitro after in vivo administration of the thymidine analogue. Full agreement between labeling pattern and corresponding DNA content was observed in pulse labeling experiments when cells submitted to mitogenic stimuli were fixed shortly after labeling, thus corroborating our observations on mouse fibroblasts in culture (Chassoux et al. 1999 ). We observed, in most fields examined, cells with a DNA content intermediate between ploidy peaks for a total level of proliferation of 13%. This indicates that proliferation can be detected by DNA content measurements alone.

Double labeling experiments suggest that alteration in the length of the cell cycle may take place at the onset of polyploidization. Delayed entry into mitosis or re-replication without mitosis has been observed in a number of biological and experimental situations. DNA damage induced either by exogenous agents, such as ionizing radiation, or by pharmacological agents results in delayed entry into mitosis to allow for repair. Mutations affecting cell cycle molecules may lead to endoreplication. Whereas both situations are unlikely to take place in physiological liver growth, disruption of specific pathways indicates possible relevant mechanisms involving, e.g., p21 (Waldman et al. 1996 ), p53 (Arora and Iversen 2000 ), and Skp2 (Nakayama et al. 2000 ). Topoisomerase II inhibition (Hasinoff et al. 2000 ), halted cyclin B1 nuclear localization (Barnes et al. 2001 ), and inactivation of membrane protein kinase (Zong et al. 2000 ) are other avenues to explore. These mechanisms can now be addressed in hepatocytes, taking advantage of fluorescence imaging.

The main advantages of in situ fluorescence imaging lies in the relationship that can be established at the single-cell level between a given biological parameter of interest (subcellular localization) and the nuclearity and ploidy, which neither flow cytometry nor cytophotometry can achieve. The precision reached can be great, providing that critical attention is paid to material preparation and to image acquisition conditions. We propose that quantitative fluorescence imaging can be used to analyze liver polyploidization mechanisms.


  Footnotes

1 These authors contributed equally to this work.


  Acknowledgments

Supported by INSERM and INRA.

We thank Catherine Senamaud–Beaufort, Helene Mouly, and Olivier Bregerie for help with cell preparations, Chantal Desdouets, Christiane Guguen–Guillouzo, and Pascale Mentré for discussions, and F. LeDiest (Hopital Necker) for FCM. We are indebted to J. Plumbridge for reading the manuscript.

Received for publication October 2, 2001; accepted October 16, 2002.


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Materials and Methods
Results
Discussion
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