Chloride turnover and ion-transporting activities of yolk-sac preparations (yolk balls) separated from Mozambique tilapia embryos and incubated in freshwater and seawater
1 Department of Anatomy, St Marianna University School of Medicine, Miyamae,
Kawasaki 216-8511, Japan
2 Department of Molecular Cell Biology, Graduate School of Medical Science,
Kyoto Prefectural University of Medicine, Kamigyo, Kyoto 602-8566,
Japan
3 Department of Aquatic Bioscience, Graduate School of Agricultural and Life
Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
* Author for correspondence (e-mail: j-hiroi{at}marianna-u.ac.jp)
Accepted 14 August 2005
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Summary |
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Key words: yolk ball, mitochondrion-rich cell, tilapia, Oreochromis mossambicus, yolk-sac membrane, chloride, turnover
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Introduction |
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Mozambique tilapia Oreochromis mossambicus is a euryhaline species
that can mature and breed in both freshwater and seawater. The embryos are
able to survive direct transfer from freshwater to seawater and vice
versa, even though the developing gills are not functional
(Ayson et al., 1994). Previous
studies have demonstrated that MRCs located in the yolk-sac membrane are the
extrabranchial site of ion exchange during the late embryonic stages of
tilapia (Ayson et al., 1994
,
1995
;
Shiraishi et al., 1997
). MRCs
in the yolk-sac membrane became larger and frequently formed multicellular
complexes when embryos were transferred from freshwater to seawater, whereas
small MRCs existed individually in freshwater
(Shiraishi et al., 1997
). Such
cellular complexes, consisting of well-developed MRCs and accessory cells, are
considered to be characteristic of seawater-type MRCs with ion-secreting
functions (Kaneko and Shiraishi,
2001
). Moreover, in vivo sequential observations on MRCs
in the yolk-sac membrane have shown that small freshwater-type MRCs are
transformed into large seawater-type cells in response to seawater transfer,
thus suggesting plasticity in ion-transporting functions of MRCs
(Hiroi et al., 1999
).
Using tilapia embryos, we have recently established a unique in
vitro experimental model, a `yolk-ball' incubation system, in which the
yolk sac is separated from the embryonic body and subjected to in
vitro incubation (Shiraishi et al.,
2001). After appropriate cutting, the incision on the yolk ball
healed during incubation in balanced salt solution (BSS), so that the yolk-sac
membrane completely enclosed the yolk. Following transfer of the yolk balls
prepared from freshwater tilapia embryos to seawater, MRCs formed new
seawater-specific multicellular complexes together with accessory cells, as
was observed in intact embryos transferred from freshwater to seawater. This
indicates that MRCs are equipped with an autonomous mechanism of functional
differentiation that is independent of embryonic endocrine and nervous
systems.
The yolk-ball incubation system would serve as an excellent experimental
model for further studies on MRC differentiation and functions. It is not
clear, however, that the ion-transporting property of the yolk balls is
comparable to that of intact embryos. In the present study, to evaluate the
ion-transporting property of the yolk balls, we examined Cl
content and turnover in the yolk balls incubated in freshwater and seawater.
To further evaluate ion-transporting functions of MRCs in the yolk-sac
membrane of the yolk balls, we investigated distributional patterns of three
ion transporters: Na+/K+-ATPase,
Na+/K+/2Cl cotransporter (NKCC), and
cystic fibrosis transmembrane conductance regulator (CFTR) that is considered
to function as apical Cl channel
(Marshall, 1995;
Singer et al., 1998
;
McCormick et al., 2003
). Our
results indicated that the yolk balls preserved the Cl
transporting property of intact embryos, and therefore help to justify the
yolk ball as an in vitro experimental model for the yolk-sac
membrane.
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Materials and methods |
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Preparation of yolk balls
The yolk balls were prepared according to Shiraishi et al.
(2001). After removing the
chorion, the embryo was placed in tilapia balanced salt solution (BSS: NaCl,
140 mmol l1; KCl, 3 mmol l1;
MgSO4, 1.25 mmol l1;
NaH2PO4, 0.4 mmol l1;
NaHCO3, 2 mmol l1; CaCl2, 1.5 mmol
l1; Hepes, 10 mmol l1; penicillin, 100 U
ml1; streptomycin, 0.1 mg ml1; pH 7.4).
The yolk sac was then cut off from the embryonic body using sterilized fine
scissors. When cut in a quick and smooth manner, the incision closed so that
the yolk material did not leak through the incision. In cases where leakage of
the yolk material was observed, the sample was discarded. After the surgical
operation, the yolk ball was incubated at 25°C in BSS for 3 h to allow the
wound to heal. The yolk-ball preparations were then incubated in freshwater
(Na+, 0.74 mmol l1; Ca2+, 0.54 mmol
l1; Mg2+, 0.26 mmol l1; pH
7.07.5) or seawater (Na+, 490 mmol l1;
Ca2+, 16 mmol l1; Mg2+, 66 mmol
l1; pH 7.58.1) for another 48 h. The incubation was
conducted in tissue culture dishes (60 mm in diameter) containing 5 ml medium
in an atmosphere of 100% air at 25°C.
Measurements of wet mass and chloride content
Wet mass of the individual yolk ball was measured to the nearest 0.1 mg
after blotting on tissues. For the measurement of Cl
content, 30 yolk balls were pooled and homogenized in 200 µl of distilled
water. The homogenate was centrifuged at 18 000 g for 15 min,
and Cl concentration of the supernatant was measured using a
chloride meter (Buchler 4-2500, Fort Lee, NJ, USA), followed by calculation of
Cl contents in the yolk balls. The chloride content was
measured in triplicate for each experimental group. The data were expressed as
the mean ± S.E.M. of total and
mass-specific Cl contents.
Chloride turnover in the yolk ball
The rate of Cl uptake was measured by whole-body influx
of 36Cl as described previously
(Miyazaki et al., 1998).
Thirty yolk balls were placed in a beaker containing 15 ml ofsterilized
freshwater or seawater, and then Na36Cl (4.37 Mbq
ml1, Amersham Biosciences, Uppsala, Sweden) was added to
give a final specific activity of 60 kBq ml1 (about 670
c.p.m. nmol1 Cl in freshwater and 2.5
c.p.m. nmol1 Cl in seawater). Changes in
Cl levels of the bathing media were negligible. At 0.5, 1,
1.5, 2, 3 and 6 h after adding Na36Cl, 5 yolk balls were removed
from the beaker using a wide-mouthed pipette, expelled into a plastic dish and
washed 5 times (1 min each) with 10 ml freshwater or seawater. The
radioactivity of the third washing solution was not different from the
background level. The samples were then placed in a miniature scintillation
vial containing 1 ml of a solubilizing agent (Soluene-350, Packard, Meriden,
CT, USA). When completely solubilized (24 h at 40°C), 3 ml scintillation
fluid (Hionic Fluor, Packard) was added and radioactivity measured using a
liquid scintillation counter (LS6000SC, Beckman, Fullerton, CA, USA).
Since the time course of 36Cl influx was
nonlinear, the rate of Cl influx was analyzed using a
first-order rate equation as described by Brown and Tytler
(1993):
Q=Qeq(1ekt), where
Q (c.p.m.) is the radioactivity at time t, Qeq
(c.p.m.) is the equilibration level of radioactivity, and k is the
rate constant (turnover rate) of influx. The turnover rate was calculated by
simple linear regression analysis of the plot of
ln(QeqQ) against t. The
Qeq (c.p.m. yolk ball1) of
36Cl was estimated by multiplying chloride
content (nmol yolk ball1) by the specific activity of the
external medium (cpm nmol1). The experiment was repeated
three times, and the turnover rate was expressed as the mean ±
S.E.M.
Triple-color whole-mount immunocytochemistry
To evaluate the ion-transporting function of MRCs, we examined distribution
patterns of Na+/K+-ATPase, NKCC and CFTR within MRCs in
the yolk-sac membrane according to the method reported by Hiroi et al.
(2005). The antibody for
Na+/K+-ATPase used here was raised against a synthetic
peptide corresponding to a highly conserved region of the
-subunit of
Na+/K+-ATPase, and has been proven to serve as a marker
for MRCs (Uchida et al.,
2000
). The affinity-purified
anti-Na+/K+-ATPase was conjugated to Alexa Fluor 546
(Katoh et al., 2003) using the Alexa Fluor Protein Labeling Kit (Molecular
Probes, Eugene, OR, USA). The antibody to detect NKCC was a mouse monoclonal
antibody directed against 310 amino acids at the carboxyl terminus of human
colonic NKCC1 (T4, developed by Christian Lytle and Bliss Forbush III;
obtained from the Developmental Studies Hybridoma Bank developed under the
auspices of the National Institute of Child Health and Human Development and
maintained by The University of Iowa, Department of Biological Sciences, Iowa
City, IA, USA). The T4 antibody has been shown to be specifically
immunoreactive to NKCC from many vertebrates including teleost fishes
(Lytle et al., 1995
;
Wilson et al., 2000
;
Pelis et al., 2001
;
Marshall et al., 2002
;
McCormick et al., 2003
;
Wu et al., 2003
;
Hiroi et al., 2005
). The
antibody for CFTR was a mouse monoclonal antibody against 104 amino acids at
the carboxyl terminus of human CFTR (R&D Systems, Boston, MA, USA). This
antibody has also been shown to detect CFTR in some teleost species
(Wilson et al., 2000
;
Marshall et al., 2002
;
Katoh and Kaneko, 2003
;
McCormick et al., 2003
;
Hiroi et al., 2005
). To allow
triple-color immunofluorescence staining, the mouse monoclonal antibodies
against NKCC and CFTR were directly labeled with Alexa Fluor 647 and Alexa
Fluor 488, respectively, using the Zenon Mouse IgG Labeling Kits (Molecular
Probes).
The yolk balls were fixed in 4% paraformaldehyde in 0.1 mol l1 phosphate buffer (pH 7.4) for 1 h, and then the yolk-sac membrane was carefully peeled off using sharp-pointed forceps. The membrane preparations were further fixed in the same fixative overnight, and preserved in 70% ethanol at 4°C. After a rinse with 0.01 mmol l1 phosphate-buffered saline containing 0.2% Triton X-100 (PBST, pH 7.2) for 1 h, the fixed yolk-sac membrane was incubated simultaneously with Alexa-Fluor labeled anti-Na+/K+-ATPase, anti-NKCC and anti-CFTR for 12 h at 4°C. Anti-Na+/K+-ATPase was diluted 1:250, and anti-NKCC and anti-CFTR were used at concentrations of 0.8 µg ml1 and 1.0 µg ml1, respectively. The antibodies were diluted with PBST containing 10% normal goat serum, 0.02% keyhole limpet hemocyanin, 0.1% bovine serum albumin and 0.01% sodium azide. The membrane was then washed in PBST for 1 h, subjected to post-staining fixation with 4% paraformaldehyde for 15 min, washed briefly in PBST, and mounted on a slide with Slow Fade Light (Molecular Probes). Confocal fluorescence images were taken using a Carl Zeiss 510 META confocal laser scanning microscope. The wavelengths of excitation and recorded emission for each Alexa dye are as follows: Alexa Flour 488, 488 nm and 505530 nm; Alexa Fluor 546, 543 nm and 560615 nm; and Alexa Fluor 647, 633 nm and 649756 nm. To avoid any crosstalk, a `multitrack' configuration was used, in which images were collected successively, rather than simultaneously, on three channels.
Scanning electron microscopic observations
To observe the apical structure of MRCs in the yolk-sac membrane, the yolk
balls incubated in freshwater and seawater were fixed in 2% paraformaldehyde +
2% glutaraldehyde in 0.1 mol l1 phosphate buffer (pH 7.4)
overnight. Subsequently, the tissues were dehydrated in ethanol, transferred
to 2-methyl-2-propanol, and dried using a freeze-drying device (JFD-300, JEOL,
Tokyo, Japan). Dried samples were mounted on specimen stubs, coated with
platinum palladium in an ion sputter (E-1030, Hitachi, Tokyo, Japan), and
examined using a Hitachi S-4500 scanning electron microscope.
Statistics
The effect of incubation medium (preincubation in BSS, and 48-h incubation
in freshwater and seawater) on wet mass of yolk balls was analyzed by a
one-way analysis of variance (ANOVA) and the TukeyKramer post
hoc test. No significant heterogeneity of variances was detected (the
Bartlett's test, P>0.05). Significant differences in
Cl content per ball, Cl content
mg1 mass and Cl turnover rates between
freshwater and seawater groups were examined using the two-sample
t-test. Data of Cl turnover rates that showed
heterogeneity of variance were square-root-transformed before the
t-test (Zar, 1999).
All analyses were conducted using JMP 5.0.1 (SAS Institute, Cary, NC, USA) and
P<0.05 was used to reject the null hypothesis.
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Results |
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Triple-color whole-mount immunocytochemistry
In the yolk-sac membrane of yolk balls incubated in freshwater,
Na+/K+-ATPase-immunoreactive MRCs were round in shape
and 1025 µm in diameter (Fig.
2A). Although a minority of MRCs in yolk balls from seawater were
of similar size and shape to MRCs from freshwater, the majority of MRCs in
yolk balls from seawater were polygonal and 2032 µm in diameter
(Fig. 2E). In MRCs of both
freshwater and seawater yolk balls, Na+/K+-ATPase
immunoreactivity was detectable throughout the cell except for the nucleus and
sub-apical region. Since it has been reported that
Na+/K+-ATPase is present on both the basolateral
membrane and the extensive tubular system that is continuous with the
basolateral membrane of MRCs (Evans et al.,
2005), the presence of Na+/K+-ATPase within
MRCs represents their basolateral distribution.
|
Enlarged images of a MRC showing basolateral Na+/K+-ATPase and apical NKCC in a freshwater yolk ball (II in Fig. 2D), and basolateral Na+/K+-ATPase, basolateral NKCC and apical CFTR in a seawater yolk ball (IV in Fig. 2H) are shown in Figs 3 and 4, respectively. In the former, NKCC staining was concentrated at the apical region of the cell (Fig. 3C,D). In the latter, CFTR immunoreactivity was detectable at the apical region (Fig. 4E,F). NKCC staining was detectable throughout the cell except for the nucleus and CFTR-positive apical region, coinciding with the staining pattern of Na+/K+-ATPase (Fig. 4AD). This cell type was accompanied by one or more accessory cells, forming a multicellular complex. The cytoplasm of the accessory cell was immunopositive for Na+/K+-ATPase but immunonegative for NKCC (Fig. 4AD). Therefore, in the triple-color-merged images (Fig. 4GI), the accessory cell remained in red, whereas the main MRC with red Na+/K+-ATPase and blue NKCC appeared in magenta.
|
|
Scanning electron microscopic observations
The apical openings of MRCs were observed by scanning electron microscopy
(Fig. 5). In both freshwater
and seawater yolk balls, most of the yolk-sac membrane was covered with
pavement cells possessing arrays of microridges. The apical openings of MRCs
were located at the boundary of the pavement cells. Although the apical
membrane was invaginated to form apical crypts in both media, the apical
openings were generally larger in seawater yolk balls than in freshwater ones.
The apical membranes of MRCs were equipped with moderately developed
microvilli in freshwater yolk balls (Fig.
5A), whereas such structures were not evident in the enlarged
apical crypt in seawater (Fig.
5B).
|
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Discussion |
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Cl concentration appears to be well regulated in the yolk
balls, because total and mass-specific Cl contents showed no
difference between freshwater and seawater yolk balls
(Table 1). However, the
Cl turnover rate measured by whole-body influx of
36Cl was about 60 times higher in yolk balls in
seawater than in freshwater (Table
1). The Cl turnover rates in the yolk balls are
comparable to those measured in intact tilapia embryos
(Miyazaki et al., 1998). The
observation of similar Cl contents of yolk balls in seawater
and freshwater suggests that the yolk balls possess mechanisms of
Cl regulation, as is the case in intact embryos. In yolk
balls incubated in seawater, where Cl permeability is
greatly increased, it is inevitable that excess Cl be
extruded in order to cope with diffusional Cl influx and
maintain the Cl concentration in the physiological range. It
would be reasonable to attribute active Cl secretion to
MRCs, which are present in the yolk-sac membrane of tilapia embryos and larvae
(Ayson et al., 1994
;
Shiraishi et al., 1997
) as
well as in that of yolk balls (Shiraishi
et al., 2001
).
On the basis of their morphology and responses to environmental salinity
changes, branchial and extrabranchial MRCs in Mozambique tilapia have largely
been classified into freshwater and seawater types
(Ayson et al., 1994;
Shiraishi et al., 1997
;
Hiroi et al., 1999
;
Uchida et al., 2000
). The
freshwater-type cells that are expected to absorb ions in hypoosmotic
environments are small and located individually. Alternatively, the
seawater-type cells, which seem to be responsible for ion secretion in
hyperosmotic environments, are larger and indented by accessory cells to form
multicellular complexes. In this study, the MRCs in yolk balls separated from
embryos were also larger in seawater than freshwater
(Fig. 2A,E) and were associated
with accessory cells when incubated in seawater
(Fig. 4GI). Our scanning
electron microscopic observations also showed a small apical pit of MRCs in
freshwater yolk balls and an enlarged pit in seawater yolk balls
(Fig. 5), which are
characteristic of freshwater-type single MRCs and seawater-type multicellular
complexes, respectively (Shiraishi et al.,
1997
). Such changes in MRC morphology are considered to reflect
alteration in their ion-transporting functions. In fact, chloride test and
X-ray microanalysis have shown that seawater-type MRC complexes have the
definitive function of Cl secretion through enlarged apical
pits, which was not observed in freshwater-type single MRCs
(Kaneko and Shiraishi, 2001
).
Taken together, the current morphological observation on yolk ball MRCs in
seawater are consistent with a function in Cl secretion.
The currently accepted model for NaCl secretion by MRCs consists of the
cooperative action of three major ion transporters:
Na+/K+-ATPase, NKCC and CFTR
(Marshall, 1995;
McCormick et al., 2003
). The
Na+/K+-ATPase, which is localized to the basolateral
membrane of MRCs, creates a low intracellular Na+ concentration and
negative charge within the cell. This Na+ gradient drives transport
of Na+, K+ and 2Cl into the cell
through basolateral NKCC. Then, Cl leaves the cells down an
electrical gradient through apical CFTR, whereas Na+ is transported
back outside the cells via Na+/K+-ATPase, and
secreted by a paracellular pathway between chloride and accessory cells.
Based on three-dimensional distribution patterns of those three
ion-transporting proteins within MRCs, we have recently reported functional
classification of MRCs in the yolk-sac membrane of Mozambique tilapia embryos
into four different types (Hiroi et al.,
2005): type I, showing only basolateral
Na+/K+-ATPase staining; type II, basolateral
Na+/K+-ATPase and apical NKCC; type III, basolateral
Na+/K+-ATPase and basolateral NKCC; and type IV,
basolateral Na+/K+-ATPase, basolateral NKCC and apical
CFTR. Type-II cells seemingly correspond to `freshwater-type' ion-absorptive
MRCs, since the cell types were not found in seawater but appeared and
increased in number after transfer from seawater to freshwater. In contrast,
type-IV cells may represents `seawater-type' ion-secretory MRCs, because the
cells were not observable in freshwater but rapidly appeared following
transfer from freshwater to seawater.
In the present triple-color whole-mount immunocytochemistry, types I, II and III MRCs were identified in freshwater yolk balls (Fig. 2D). It is likely that freshwater yolk balls preserved the MRC populations of the intact embryos that had been kept in freshwater, being favorable for freshwater acclimation. Alternatively, the yolk balls incubated in seawater were characterized by the predominant occurrence of type-IV cells with basolateral Na+/K+-ATPase, basolateral NKCC and apical CFTR. The intracellular localization of those three proteins within type-IV cells is completely consistent with the current accepted model for ion secretion by MRCs, and the predominant occurrence of type-IV cells can account for active ion secretion in seawater yolk balls.
Type-IV MRCs were not observable in intact embryos reared in freshwater,
but rapidly appeared following transfer from freshwater to seawater; these
type-IV cells were previously thought to originate mostly from pre-existing
type-I cells and type-III cells (Hiroi et
al., 2005). Type-IV cells observed in seawater yolk balls are also
considered to develop from pre-existing type-I and type-III cells after
exposure to seawater. This conversion of MRC types in the yolk-sac membrane of
yolk balls, which reflects changes in the expression and localization of ion
transport proteins, is likely to be independent of the embryonic endocrine and
nervous systems. Similarly, it has been demonstrated in killifish that
seawater-type MRCs are transformed into freshwater-type cells as a short-term
response to transfer from seawater to freshwater, followed by the promotion of
MRC replacement as a long-term response
(Katoh and Kaneko, 2003
).
Although we did not address Na+ permeability in yolk balls,
Na+ influx is expected to be higher in seawater than in freshwater,
as seen in Cl permeability. Occurrence of
Na+/K+-ATPase in the basolateral membrane of type-IV
MRCs in seawater yolk balls implies a Na+-secreting function. This
is also supported by the formation of multicellular complexes consisting of
type-IV and accessory cells, presumably providing a paracellular pathway for
Na+ secretion (Shiraishi et
al., 1997).
In vitro experimental models have been used for MRC research in
the past, such as primary cultures of gill epithelial cells
(Battram et al., 1991;
Perry and Walsh, 1989
;
Pärt and Bergström,
1995
; Avella and Ehrenfeld,
1997
; Wood and Pärt,
1997
), and opercular and yolk-sac membrane preparations
(Karnaky et al., 1977
;
McCormick, 1990
;
Marshall, 1995
;
Ayson et al., 1995
). The
yolk-ball incubation system that we have established has the following
advantages over the conventional in vitro experimental models: (1)
the tissue including MRCs can survive for a long period, so that the
morphological alteration of MRCs can be followed; (2) MRCs maintain the
ability of cellular differentiation; (3) the tissue preparation is free from
the embryonic endocrine and nerve systems; and (4) the cellular polarity can
be maintained and the serosal side of the yolk-sac membrane is separated from
the external environment (Shiraishi et
al., 2001
). In addition, the present study has demonstrated that
the yolk ball preserves the Cl transporting properties of
the intact embryo, presumably because the yolk-ball incubation system utilizes
the intact yolk-sac membrane of tilapia embryos without cellular
reconstitution. These findings help establish the isolated yolk ball as an
in vitro experimental model for the yolk-sac membrane containing
MRCs. The ion-absorptive functions by freshwater-type MRCs are currently less
well understood than seawater-type ion-secretory MRCs
(Perry 1997
;
Evans et al., 2005
). In
addition to molecular cloning and immunocytochemical studies on ion
transporters that could be involved in ion uptake from freshwater, such as
vacuolar-type H+ ATPase, Na+ channel,
Na+/H+ exchanger and NKCC, yolk balls are expected to
serve as a suitable model to examine the ion-uptake functions of these
transporters.
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