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Department of Zoology, The University of Hong Kong, Hong Kong
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of the present study was to characterize chloride cell subtypes in the fish gill and to monitor the kinetic change of cell division in the gill epithelia during seawater adaptation. Employing a three-step Percoll gradient method, the gill chloride cells and nonchloride cell population were isolated. The isolated cells were studied using multiparameter flow cytometry, recording the changes in 1) cell size, 2) cellular granularity, and 3) cell autofluorescence. Two chloride cell subtypes were identified in the freshwater eels. Within 2-4 days after entering seawater, new subtypes of transitory chloride cell, with bigger cell size and more intense mitochondria autofluorescence, appeared. After full adaptation, two major seawater chloride cell subtypes were again discerned; their sizes were the largest and their mitochondria autofluorescence was the highest. In the second part of the experiment, cell cycle analysis demonstrated a progressive increase in the percentage of gill cells entering the DNA synthesis phase during seawater adaptation, where a small population of mitotic cells was identified in the nonchloride cell population but not in chloride cells. We hypothesize that the mitotic cells identified are stem cells, which will ultimately differentiate into seawater chloride cells. Our results confirm the existence of heterogeneity of chloride cells. Individual subtypes could be isolated in high purity for further studies to elucidate their respective function in mediating ion transport.
flow cytometer; confocal microscope; pavement cell; DNA cell cycle; sorting
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE GILL OF TELEOST FISH, the site for active ion transport, contains chloride cells, pavement cells, and other epithelial cells. The chloride cell is a mitochondria-rich ionocyte, which plays a crucial role in osmoionoregulatory function. After seawater transfer, there was an increase in the mitotic activities of the gill epithelia (4-6, 17) followed by the development of highly differentiated chloride cells (8-10, 14, 20). The existence of heterogeneity of chloride cell subtypes has been demonstrated in the past with its corresponding salinity-related changes in cell number and/or size, mitochondria content, and Na+-K+-ATPase activities reported (12, 14, 15). However, the underlying process is still not fully elucidated. Most studies were performed on the basis of histological approach. The identified cell types could not be further characterized, and the staining procedure could itself induce changes in the cell morphology, creating some difficulties in cell subtype identification (16). Nevertheless, only a few data are available in the literature concerning the kinetic changes of gill cells during seawater adaptation. Hence, the important issue as to understanding the cytological and kinetic changes of the gill cells and their respective functions was left unresolved. The identification of the origin and of the subtypes of the chloride cells was the subject of this study.
Our success in isolating viable pavement and chloride cells from the gill epithelia of the eels (20) provides an excellent opportunity to study the cytological and kinetic changes of these cells after transfer from freshwater to seawater. To avoid the above-mentioned problems associated with histological analysis, we recently developed a rapid procedure using flow cytometry for the investigation of gill cell cytological change on seawater adaptation. In this study, we further extended the procedure to enable the simultaneous measurement of the changes in cell size, granularity, and autofluorescence. Furthermore, by labeling the cells with a DNA stain, e.g., propidium iodide, univariate DNA analysis provided reliable estimates of the cell cycle kinetics.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Japanese eels (Anguilla japonica) weighing between 500 and 600 g were reared in a fiberglass tank supplied with charcoal-filtered aerated tap water at 18-20°C under a 12:12-h light-dark photoperiod for at least 3 wk. Because the fish were sexually immature (gonadosomatic index
0.18%), all of mixed sexes were used to prepare
gill cells for this study. The fish were transferred to 50% seawater
and then to full seawater, and they were killed at predetermined
intervals from 1 day to 2 wk.
Cell Isolation
The eel was anesthetized, and the gill cells were isolated using a three-step discontinuous Percoll gradient system as already described (20). Briefly, gills were perfused with a buffered saline (in mM: 130 NaCl, 2.5 KCl, 5 NaHCO3, 2.5 glucose, 2 EDTA, 10 HEPES, pH 7.0) to remove blood cells. Gill arches were excised and washed. Epithelia were scraped off from underlying cartilage with a glass slide into Ca2+-, Mg2+-free Hank's balanced salt solution (HBSS, Sigma). The scrapings were dispersed by being passed through two stainless steel filters with mesh size of 104 and 73.7 µm, respectively. It was then digested by 1.25 mg/ml collagenase (Sigma Type IA) and 2 mg/ml hyaluronidase (Sigma Type I-S) at room temperature for 30-45 min. The cell suspension was washed by Ca2+-, Mg2+-free HBSS and finally resuspended in 1.06 g/ml Percoll solution. A three-step gradient, 1.09, 1.06, and 1.03 g/ml, was prepared and was centrifuged at 2,000 g, 15°C, for 15-20 min. Isolated cell fractions were concentrated by centrifugation, and the pellet was processed for mitochondria staining and scanning electron microscopic examination as described previously (20). Pavement cells showed characteristic microridges on their cell surface, whereas chloride cells demonstrated apical pits. In addition, chloride cells were further verified by intense mitochondria staining.Flow Cytometric Analysis of Gill Cells
Flow cytometric analysis was performed on a Coulter EPICS Elite Flow Cytometer (FCM) equipped with an Argon laser (15 mW) emitting at 488 nm. The instrument had a standard optical filter configuration with band pass 530/30 nm and 585/42 nm filters for FL1 and FL2, respectively, and a 610-nm band pass filter for FL3. The FCM was calibrated before use with standard DNA beads. Thirty eels were used in this experiment.Isolated gill cells (~107 cells/ml in HBSS) were analyzed directly by FCM. The change in cell size [forward light scatter (FLS)], granularity [side light scatter (SLS)], and autofluorescence at 525 nm (FL1) were recorded. Individual cell populations were sorted to reconfirm the cell type identification using mitochondria staining. Briefly, the sorted cells were stained by incubation with 1 mM rhodamine-123 for ~45 min. After washing in 10 mM PBS, the preparation was examined (excitation 488 nm, emission 512 nm) in a laser confocal microscope using an Argon light source (BioRad, MRC-600).
DNA Cell Cycle Analysis
The isolated suspended cells, at a concentration of 2 × 105/ml, were fixed by the vigorous addition of 5 ml ice-cold 70% ethanol and stored at 20°C for no more than 1 mo. Before analysis, the cells were centrifuged at 1,000 g for 5 min, washed twice with cold PBS, and finally resuspended in 0.8 ml PBS, ensuring that a single cell suspension was obtained. DNA staining and RNA digestion was achieved by the addition of 0.1 ml 400 µg/ml propidium iodide (Sigma) and 0.1 ml 2 mg/ml RNase (Sigma), respectively, and incubated at 37°C for 30 min in dark. The cells were then stored at 4°C overnight before analysis. Cellular DNA content was determined by FCM with detection through FL3.Statistical Analysis
All data are represented as means ± SE. Statistical significance is tested by Student's t-test and ANOVA depending on the number of means. The level of significance is set at P value of < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated Gill Cell Analysis
FLS, SLS, and FL1 values for the gill cells were used to monitor the change in cell size, granularity, and autofluorescence before and after seawater transfer. FCM analysis distinguished between chloride and nonchloride cells on the basis of the bivariate FLS/FL1 or SLS/FL1 analysis. Figure 1 showed the FLS/FL1 analysis of nonchloride cells and red blood cells. The nonchloride cells had lower autofluorescence compared with chloride cells (Fig. 2). The bivariant histograms given in Fig. 2, A-D, (FLS/FL1) and Fig. 3, A-D, (SLS/FL1) showed the progressive change of the chloride cell subtypes from freshwater to 2 days 50% seawater, 3 days 50% seawater, and seawater conditions. Throughout seawater transfer, there was no change in nonchloride cells in terms of cell size, granularity, and autofluorescence.
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Bivariant Cell Size/Autofluorescence Analysis
In freshwater-adapted eel (Fig. 2A), two chloride cell subtypes were identified and are defined here as F1 and F2 cells (F = freshwater). The F1 cells were bigger in size, but contained relatively lower levels of autofluorescence than F2. The relative proportions of these two types of chloride cell populations were 67.5 ± 4.2 and 32.5 ± 4.2% for F1 and F2, respectively.After a 2-day adaptation in 50% seawater (Fig. 2B), two new chloride cell subtypes appeared and are defined here as transitory cells (T1 and T2). Sizes of these cells were bigger than those of the F1 and F2 cells and the level of autofluorescence was higher than that of the F1 cell. The relative percentage of F1 cells decreased from 67.5 ± 4.2 to ~29 ± 3.4%, and the F2 cells remained at ~35.5 ± 3.8%. The newly appeared T1 and T2 subtype populations amounted to 11.2 ± 2.1 and 24.3 ± 1.6%, respectively. T2 cells had higher autofluorescence than T1 cells. After a 3-day adaptation in 50% seawater (Fig. 2C), F1 totally disappeared and the F2 subtype decreased to ~24%. Meanwhile, T1 and T2 cell populations increased to ~12.2 ± 2.4 and 63.8 ± 3.3%, respectively.
After a 2-wk seawater acclimation (Fig. 2D), cells with the FLS/FL1 characteristics of the T1 and T2 cells were no longer present, but seawater chloride cell subtypes, called S1 and S2, with different characteristics were found. The S1 and S2 cells had the biggest cell size and highest autofluorescence among all, with the relative proportion of the S1 and S2 cell populations amounting to 48.8 ± 3.1 and 51.2 ± 3.1%. respectively. S2 cells had higher autofluorescence than S1 cells.
Bivariant Cellular Granularity/ Autofluorescence Analyses
Figure 3, A-D, using SLS/FL1 shows the progressive changes of two freshwater chloride cell subtypes into seawater chloride cells, which contain the highest granularity and autofluorescence. Transitory chloride cells were hardly identified.Analysis of Cellular DNA Using Propidium Iodide Staining
Nonchloride cell. As noted in the univariate DNA analysis (Fig. 4), the nonchloride cell population was predominantly diploid. In all the samples analyzed in this study, the coefficient of variation of the DNA histogram, derived from the isolated nonchloride cell stained with propidium iodide, ranged between 2 and 6%. Estimates of cell cycle distribution on the basis of univariate DNA analysis are summarized in Table 1. There appeared to be a slight shift in the phase distribution after 24-h seawater adaptation, where the number of cells in G0/G1 phase decreased from 84.20 to 82.60%, concomitant with significant (P < 0.05) accumulation of S phase cells, from 4.53 to 6.98%, compared with the control. No significant alternation in the G2/M phase cell distribution was detected. After 48-144 h, there was significant (P < 0.05) increase of the cells in S phase (6.98-17.18%), together with significant (P < 0.05) reduction in G0/G1 phase (82.60-72.70%). In seawater, the relative percentage of the cell cycle stages were readjusted to G0/G1 = 75.50%, S phase = 10.83%, and G2M phase = 13.60%.
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Two separate gatings (A and
B) were set on the nonchloride cell
population (Fig.
5A) on
the basis of the FLS and SLS parameters. The individual gated cell
population was reanalyzed to produce two separate DNA histograms (Fig.
5, B and
C). The results demonstrated that
mitotic activities were primarily recorded in the bigger cell
population (gate
A), whereas the smaller cell
population (gate
B) was predominantly in diploid
stage.
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Chloride cell. The results from DNA histograms suggested that seawater transfer had no effect on the phase distribution of chloride cells. Profiles from seawater-adapted fish chloride cells were similar to the control after the transfer. In the univariate DNA analysis of the isolated chloride cells, they were all in diploid stage. No S phase or G2/M phase appeared throughout the adaptation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, FCM provided an appropriate and sensitive approach to monitor the cytological changes of the isolated chloride cells and the nonchloride cell population [consists of pavement cells: ~96%, undifferentiated cells and cells of the basal layer in the lamellar epithelium (20)] during seawater transfer. Three parameters [FLS, SLS, and autofluorescence at 525 nm (FL1)] were employed to measure the cellular parameters, relative cell size, cellular granularity, and autofluorescence, respectively, arisen from their corresponding cellular constituents. FL1, in the present study, detected autofluorescence, probably originated from the intracellular constituents (i.e., NADH, riboflavin, flavin coenzymes, and flavoprotein) bound in the mitochondria (1). Therefore, the autofluorescence intensity detected may reflect the intracellular concentration of these compounds and hence the quantity or activities of the mitochondria in the cells. In the past studies, ultrastructural analysis had counted on the acid fuchsin (15), metallic impregnation (10, 12), or immunohistochemical (14) staining on tissue sections. In the present study, no staining was required to identify different cell types. The cellular parameters collected by FCM during data acquisition reflected the changes in the size, granularity, and autofluorescence of the living cells.
The first study concerning chloride cell subtypes in gill epithelia was
based on the ultrastructural appearance, in which chloride cells had
been categorized into two types: electron dense and electron light (7).
However, Straus (16) was not convinced of their separate identity and
observed that most cells were intermediate between the two extremes of
cytoplasmic densities. The various observations cast a doubt as to the
existence of heterogeneity of chloride cell subtypes in the gills.
Alternative criteria on distinguishing the two chloride cell subtypes
were then demonstrated according to their shape and location in the
primary and secondary gill epithelia (2). Then using acid fuchsin
stain, Shirai and Utida (15) described a strongly acidophilic A-type
(strong mitochondria staining with smaller size) and weakly acidophilic
(weak mitochondria staining with bigger size) B-type chloride cell in
the gill epithelium of the Japanese eel. Pisam and Rambourg (12)
renamed these cells as 
-cell and 
-cell. The -cell was pale and
elongated under the electron microscope, located at the base of the
secondary lamellae. The -cell was darker and ovoid in shape and was
located in the interlamellar region. In the present study, chloride
cell subtypes were identified, in agreement with the results obtained in previous observations. Comparison between FLS/FL1 and SLS/FL1 analysis showed the former to be more effective in chloride cell subtype identification. It indicated that changes in cell size and
cellular autofluorescence (mitochondria content) were distinctive cytological parameters during the development of the seawater chloride
cells (20). Therefore, in the present report, the description of
chloride cell subtypes was based on the FLS/FL1 analysis. In the
seawater transfer experiment, "freshwater," "transitory," and "seawater" type chloride cells were identified. Presumably, freshwater cells developed into the transitory type before developing into the seawater type. The development involved an increase in cell
size, granularity, and the intensity of mitochondria autofluorescence. The F1 cell was the main subtype (67.5%) in freshwater fish gill, but
the percentage decreased on seawater adaptation on
day 2 (29%) until it totally disappeared on
day
3. The percentage of F2 subtype remained the same on the first 2 days but decreased to 24.3% on day
3. In comparison to the previous
findings, the F1 and F2 subtypes were similar to the B ( type) and A
( type) chloride cells, respectively (12, 15). The relative
percentage of the two cell subtype populations was similar to that
described by Utida et al. (18). The F1 subtype was larger with lower
mitochondria autofluorescence, whereas the F2 subtype was smaller but
with higher mitochondria autofluorescence. It seemed that F1 gave a faster response to the initial phase of seawater adaptation than F2
cells, indicating its importance in freshwater environment, as
described by Pisam et al. (11) and Shikano and Fujio (14), where
-cell was specifically involved in freshwater adaptation. Shirai and
Utida (15) described a decreased percentage of B cells followed by an
increase in A cells during seawater transfer. However, there was no
appearance of any transitory type of chloride cell. Similar findings
were obtained by Pisam and Rambourg (12) and Shikano and Fujio (14),
but with the appearance of the transitory type of chloride cell
reported. Our findings in seawater transfer confirmed the disappearance
of the F1 subtype on day
3 transfer (19) and the emergence of
two transitory chloride cell subtypes (T1 and T2) that finally
developed into seawater chloride cells. It seemed that seawater
chloride cells were developed from the differentiation of F2, T1, and
T2 chloride cell subtypes. It is suggested that most of the F2 subtype
was able to transform into seawater chloride cells, as in the case of
-cells, whereas the F1 subtype degenerated during the transfer
(12-15). However, no explanation can be provided as to why the
number of chloride cells in the seawater condition increased by 10-fold
(10, 15, 18, 20). A better understanding on the origin of the seawater
chloride cell depends on direct measurement of gill mitotic activities.
There have been some studies demonstrating an increase in mitotic activity in the gill epithelium after transfer of fish from freshwater to seawater (6). The newly differentiated cells were found to be located in the interlamellar region of the filament and were suggested to be chloride cells. Seawater-induced synthesis of DNA in the gill epithelia was confirmed later in eels (3, 4, 17) and in Lebistes reticulatus (5). In the studies in eels, enzymes associated with the synthesis of carbomylphosphate (a precursor of pyrimidine) were elevated before DNA synthesis followed by cellular reorganization in the gill epithelium. In the latter study, a greater turnover of undifferentiated cells in seawater was reported in association with the development of chloride cells in the deeper cell layers of primary epithelium from undifferentiated cells. The present study demonstrated that seawater transfer did not cause any effect on the phase distribution of chloride cells, thus ruling out the possibility of freshwater chloride cells dividing into seawater chloride cells. To identify the mitotic cells, two gates were set separately on the histogram showing the nonchloride cell population. The position of the gates was determined by the cell size. The gated populations produced independent DNA histograms in which different mitotic activities could be calculated. The mitotic cell had a bigger cell size, which can be interpreted as necessary to house the newly synthesized genetic and cytoplasmic materials for two daughter cells. Most of newly synthesized daughter cells could differentiate into seawater chloride cells. Clearly, our findings are consistent with the previous studies (5, 17) demonstrating the origin of chloride cell arising from the stem cell population. In addition, we provided further evidence for the kinetic change of the cells.
In conclusion, we have shown the particularities of the chloride cell subtypes in gill epithelia of the eels adapting in different salinities, indicating that substantial changes in cell size and autofluorescence occur during seawater transfer. The corresponding increase in the seawater chloride cell population was derived from the differentiation of existing freshwater (F2), transitory (T1, T2) chloride cells and the newly synthesized daughter cells. The present study provides a new approach for the investigation of the cytological and kinetic changes of the gill cells. The identified nonchloride cell population and different chloride cell subtypes could be isolated for further investigation and characterization.
Perspectives
The techniques described here, together with the cell isolation protocols (20), provide a useful tool to detect and monitor the cytological change of the gill cells under numerous physiological challenges (i.e., acid/alkaline stress, hypoxia, and chemical toxicity). The limitation encountered, however, is the inability to correlate the particular chloride cell subtypes to their respective locations in gill epithelia. This could be overcome with the use of specific cell markers to obtain locations of the isolated cells; yet these markers are not currently available. The merit of the technique used in this study is its ability to provide good statistics in high numbers of cell analysis and to isolate individual chloride cell subtypes in high purity. Differential gene expression analysis can then be undertaken to understand their respective roles. Our next step is to study the hormone receptor (i.e., stanniocalcin, cortisol, urotensins, or natriuretic peptides) interaction by measuring the changes in the intracellular Ca2+, Na+, and pH of the gill cells (4). Indeed, it could be further extended to determine the specific response of particular chloride cell subtype by using the gating function of the FCM.| |
ACKNOWLEDGEMENTS |
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This work was supported by the Research Grants Council, Hong Kong.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. K. C. Wong, Dept. of Biology, Hong Kong Baptist Univ., Kowloon Tong, Hong Kong (E-mail: ckcwong{at}hkbu.edu.hk).
Received 20 November 1998; accepted in final form 26 April 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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