Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills
1 Division of General Physiology, Department of Biology, University of Oslo,
PO Box 1051, 0316 Oslo, Norway
2 Institute of Pathology, Norwegian National Hospital, Oslo,
Norway
* Author for correspondence (e-mail: jorund.sollid{at}bio.uio.no)
Accepted 11 July 2003
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Summary |
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Key words: crucian carp, Carassius carassius, gill, secondary lamellae, morphology, hypoxia, apoptosis
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Introduction |
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The crucian carp (Carassius carassius) is a North European
freshwater fish that often inhabits small ponds that, due to ice coverage,
become hypoxic and finally anoxic for several months every winter
(Holopainen et al., 1986). Its
exceptional hypoxia and anoxia tolerance make it the sole fish species in this
habitat. In both Norwegian and Swedish populations of this carp, we have
observed a lack of protruding lamellae, the respiratory units in fish
(sometimes denoted secondary lamellae). This is a highly exceptional feature
in fish since the lamellae are the primary site for gas exchange, making up
most of the respiratory surface area of fish gills. However, a favourable
aspect of a small gill area would be reduced water influx and ion losses.
The haemoglobin of Carassius has an extremely high affinity for
O2; a P50 for O2 of 0.347 kPa has
been measured in goldfish (Carassius auratus;
Burggren, 1982). This may
explain why, under normoxic conditions, crucian carp do not need a large
respiratory surface area. Thus, the lack of protruding lamellae could be a
morphological adaptation for reducing water and ion fluxes. However, such an
unusual trait should limit the ability of the crucian carp to cope with
falling environmental O2 conditions, unless relatively rapid
morphological changes can take place.
Here, we present evidence that such a change is actually taking place in the gills of crucian carp when exposed to severe hypoxia. The change is reversible, and we suggest that this plasticity is an adaptive mechanism that promotes aerobic metabolism under hypoxic conditions and reduces problems related to osmoregulation under normoxic conditions.
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Materials and methods |
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Hypoxia exposure
During hypoxia exposure, the fish were kept in dark 25-litre plastic tanks
continuously supplied with N2-bubbled dechlorinated Oslo tap water.
The oxygen level was monitored with an oxygen electrode (WTW, Weilheim,
Germany), which also controlled a valve that varied the rate of N2
bubbling according to the desired level of hypoxia. The water temperature was
8°C. The fish were exposed to hypoxia for up to 14 days. Fish were taken
out for sampling at 0, 1, 3, 7 and 14 days of exposure. The O2
level was kept at 0.75±0.15 mg l-1 (6-8% of air saturation).
This is slightly below their critical O2 concentration
([O2]crit), which is the level of O2 where
the fish can no longer meet their energy requirements through aerobic
metabolism alone (Beamish,
1964). [O2]crit of crucian carp is
approximately 1.0 mg l-1 at 8°C
(Nilsson, 1992
). A group of
fish were put back into normoxia for 7 days after 14 days of hypoxia exposure
and were then sampled. Control fish (and reoxygenated fish) were kept in an
identical tank, which was supplied with aerated water rather than
N2-bubbled water. [O2] in this tank was 10-11 mg
l-1. The hypoxia experiments where done from March to October 2000
and 2001.
Scanning and transmission electron microscopy (SEM and TEM)
Gills were fixed in 3% glutaraldehyde in 0.1 mol l-1
Nacacodylate buffer. For SEM, the gills were dehydrated and dried with Blazers
Critical Point Drier (Hypervision, Fremont, USA) before being AuPd coated with
an SEM coating unit (E5000; Polaron Equipment Limited, Watford, UK). For TEM,
the gills were post-fixated in 2% osmium in 0.1 mol l-1
Nacacodylate buffer with 1.5% potassium-ferri-cyanide before en bloc
incubation in 1.5% uranylacetate. The tissue samples were then dehydrated
before being embedded in Epoxy embedding medium (Fluka, Buchs, Switzerland).
The microscopes used were a JSM 6400 from JEOL (Peabody, USA) for SEM and a
CM100 from Phillips (Potomac, USA) for TEM.
Morphometry
The area of the portion of the lamellae in contact with water was
calculated. To do this, three measurements were taken on randomly selected
gill filaments from four normoxic and four hypoxic fish (see
Fig. 1; lowercase letters
denote measurements in normoxia, and uppercase letters denote measurements in
hypoxia): (1) the basal length of the part of the lamellae in contact with
water was measured using SEM (l and L, respectively); (2)
the mean height of the lamellae in contact with water was measured using light
microscopy on the sections obtained in the BrdU and TUNEL experiments (see
below); the latter was done on cross-sections where one-third of the
measurements were done when the central venous sinus was visible and
two-thirds were done when it was not; thus, one-third were from the central
portion of the lamellae (hc and Hc,
respectively) and two-thirds were from the edges (he and
He, respectively); (3) the mean lamellae thickness
(t) was also measured using light microscopy on BrdU and TUNEL
sections.
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The area of the normoxic lamella (a) was approximated by
calculating the lamellar tip area, a=pl, where l is
the protruding basal length and p is the ellipse perimeter formula
divided by two:
![]() | (1) |
The total lamellar area in hypoxia (A) was approximated by
multiplying the mean protruding height minus the mean height of the lamella
tip (obtained in the measurements of normoxic lamellae) by the mean basal
length and finally adding the area of the lamellar tip:
![]() | (2) |
![]() | (3) |
Note that most histological procedures are likely to distort the tissue, and comparisons between studies should be made with caution.
Respirometry
Fish kept in normoxia (with embedded lamellae) were compared with fish
exposed to hypoxia (with protruding lamellae). The latter group had been
exposed to hypoxia for 7 days and then kept in normoxic water for 24 h to
minimise the possibility that the fish suffered from hypoxia-induced energy
deficiency, even if this is unlikely for crucian carp. The rate of
O2 consumption
(O2) during
falling water [O2] was measured with closed respirometry, and the
[O2]crit was determined as described previously
(Nilsson, 1992
).
High salinity exposure
Fish with embedded and protruding lamellae were obtained as described above
for the respiration experiment. Fish were then subjected to normoxic saline
waters (containing 50% seawater and 50% Oslo tap water, giving a salinity of
16 p.p.m.) for 0, 1.5 and 6 h. Blood samples were taken from the caudal
vessels by heparinised 1 ml syringes. The blood plasma chloride content was
analysed using a CMT10 Chloride Titrator (Radiometer, Lyon, France).
Measurement of mitotically active cells by 5'-bromodeoxyuridine
(BrdU)
BrdU has previously been successfully used to stain mitotically active
cells (cells in the S phase) in fish gills
(Tsai and Hwang, 1998;
Uchida and Kaneko, 1996
). BrdU
was injected intraperitoneally at a dose of 100 µg g-1. After 24
h, the fish were killed with a sharp blow to the head, and the gills were
dissected out, fixated in 4% formaldehyde in 0.1 mol l-1
phosphate-buffered saline (PBS) and embedded in liquid paraffin before being
serially cross-sectioned (thickness, 2 µm). One slide from each fish was
rehydrated before being placed in 2 mol l-1 HCl for 30 min. They
were quenched in 3% H2O2 and subsequently incubated for
30 min in 5% blocking solution (denoted BS; 5% bovine serum albumin and 0.3%
Triton X-100 in PBS, pH 7.2). 65 µl of anti-BrdU mouse IgG, diluted 1:5 in
0.5% BS (BS diluted 10x with PBS, pH 7.2) were applied and incubated for
1 h in a humidified chamber. 70 µl of biotinylated goat anti-mouse IgG,
diluted 1:20 in 0.5% BS, were applied and incubated for 30 min in a humidified
chamber. 70 µl of peroxidase conjugate, diluted 1:20 in 0.5% BS, were
applied and incubated for 30 min in a humidified chamber. The slides were
washed three times with PBS (pH 7.2). 65 µl of diaminobenzidine (DAB)
solution (6 mg DAB, 10 ml PBS and 30 µl 30% H2O2)
were then applied for 3-10 min before the slides were washed, put briefly (3-5
s) in haematoxylin and mounted. Between all the steps, from placing in 2 mol
l-1 HCl to incubating with peroxidase conjugate, a double wash with
PBS/saponin (1 g saponin:l ml PBS) was used. All solutes were from
Sigma-Aldrich (St Louis, USA), except anti-BrdU (Becton Dickinson, Franklin
Lakes, USA). Sections from the gastrointestinal tract of the crucian carp were
used as positive controls. The staining pattern agreed well with what is known
about gastrointestinal cell proliferation in the tract (not shown). As a
negative control, BrdU-containing sections from normoxic fish were used. The
sections were incubated without anti-BrdU. The pre- and post-treatments were
conducted as above. This test did not show any non-specific binding of the
secondary or tertiary antibodies.
Detection of apoptotic cells using TdT-dUTP-nick end labelling
(TUNEL)
An ApopTag®Plus Peroxidase In Situ apoptosis detection kit
from Intergen (Temecula, USA) was used. No modifications of the protocol
included in the kit were necessary. The gills were dissected, fixed, embedded
and sectioned as described in the BrdU method. The sections were
counterstained with haematoxylin before being mounted. In the rodent mammary
gland, extensive apoptosis occurs 3-5 days after weaning of rat pups. Sections
of this tissue were included in the kit and were used as positive controls.
1-2% of the total number of cells on the slides was apoptotic, as predicted in
the kit instructions. A section from the mammary gland and a section from the
gills were used as negative (substitution) controls. This was done by
excluding the TdT but including the proteinase K digestion to control for
non-specific incorporation of nucleotides or for non-specific binding of
enzyme conjugate. Equilibration buffer was substituted for the volume of TdT
enzyme reagent. No stained cells were seen in the negative controls. The TUNEL
method distinguishes apoptosis from necrosis by specifically detecting DNA
cleavage and chromatin condensation associated with apoptosis. However, there
were some instances where cells exhibiting necrotic morphology were diffusely
stained. Therefore, stained cells were only counted if they showed
morphological criteria consistent with apoptosis (nuclear condensation, cell
shrinkage and apoptotic bodies). This was assessed using light microscopy.
Initially, TEM was used to confirm apoptosis morphologically
(Fig. 2) in a few sections. The
number of stained cells without apoptotic morphology was below 1% of the total
number of stained gill cells in hypoxia-exposed fish, clearly indicating that
apoptosis, and not necrosis, is involved in reducing the ILCM.
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Counting of cells
Stained (BrdU and TUNEL) and non-stained cells in the ILCM (all cells
between two adjacent lamellae) were counted in randomly selected areas on the
sections by two persons to a total number of 500-600 cells per fish in all
immunohistochemical analyses, giving between 15 and 25 ILCMs per fish.
Statistics
All data were analysed with a non-parametric Kruskal-Wallis test with
Dunn's test as a post-test, except for morphometry, which was analysed with an
unpaired Student's t-test. The data were analysed by the program
GraphPad InStat (GraphPad, San Diego, USA).
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Results and discussion |
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Morphometry
Light microscopic examination of serially cross-sectioned gill arches
showed that the lamellae were also present in normoxia but that the space
between the lamellae was then completely filled by an interlamellar cell mass
(ILCM). After 7 days in hypoxia, the mean area of the portion of the lamellae
that was in contact with water increased by 7.5-fold, from 1195
µm2 per lamella in normoxia to 8898 µm2 per
lamella in hypoxia (Table 1).
This resulted from the reduction of the ILCM, which was reduced by
52%
from 2.01x105 µm3 per interlamellar space in
normoxia to 9.79x104 µm3 in hypoxia.
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The reduction of the ILCM and the resultant exposure of a much larger
lamellar area to water will greatly increase the part of oxygen transfer aided
by convective water movements. However, some oxygen can be expected to diffuse
through the ILCM to reach the blood inside the lamellae. We do not know how
important this route is but it is likely to contribute to some of the oxygen
uptake, especially in carp with embedded lamellae. However, in those fish, it
is possible that most of the O2 is taken up by erythrocytes passing
through the outer marginal channel, which runs immediately inside the edge of
the lamellae of fish gills (Laurent and
Dunel, 1980). In the carp with embedded lamellae, about half of
the lamellae had their outer edges not covered by ILCM cells. Fishes appear to
have the ability to redirect blood flow from the lamellar sheet to the outer
marginal channel (Stensløkken et
al., 1999
) and such a mechanism could be in operation in carp with
embedded lamellae. Still, the increased lamellar area must significantly
increase the capacity for oxygen uptake, and it is also likely to increase
water and ion fluxes between water and blood. Indeed, such effects were
clearly indicated by the subsequent experiments described below.
Respiration
We hypothesised that this large increase in the respiratory surface area
was beneficial for the ability to take up O2 in hypoxia. Indeed,
our closed respirometry experiment showed that the group with protruding
lamellae had a 50% lower [O2]crit (0.5±0.1 mg
l-1) compared with that of the group with embedded lamellae
(1.0±0.1 mg l-1; P<0.01;
Table 2). The normoxic rate of
O2 consumption
(O2) was
96±8 mg kg-1 h-1 in fish with embedded lamellae
and 84±7 mg kg-1 h-1 in fish with protruding
lamellae. These rates were not significantly different (P>0.05),
which indicates that the difference in [O2]crit was not
caused by differences in
O2. Thus, we
attribute the lower [O2]crit in fish with protruding
lamellae to their larger respiratory surface area. However, the presumed
increase in costs for osmoregulation in crucian carp with protruding lamellae
was apparently not large enough to be detected in this experiment. This may
relate to the relatively large variability in
O2.
Osmoregulatory costs in fish are thought to account for approximately 10% of
the energy budget (Boeuf and Payan,
2001
), and a reduction or increase in this figure may therefore
not be readily detected with respirometry. Another possibility is that the
fish recently exposed to hypoxia are metabolically depressed, and this could
mask an increase in
O2 related to
increased osmoregulation costs.
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Our data indicate that the crucian carp can take advantage of the very high
ability of its haemoglobin to extract oxygen in such a way that it can reduce
its gill surface under normoxic conditions without depressing
O2 and, in this
way, reduce osmoregulatory costs.
Since the crucian carp is an exceptionally anoxia-tolerant fish, one may
argue that it would not need to increase its capacity for oxygen uptake when
faced with hypoxia. However, in anoxia, crucian carp produce ethanol as the
main anaerobic end product (Nilsson,
2001). The ethanol is lost to the water, making this an
energetically wasteful strategy. Thus, it should be advantageous for it to
avoid utilising anaerobic ATP production for as long as possible, particularly
since its survival during anoxia is ultimately limited by the total depletion
of its glycogen stores (Nilsson,
1990
).
There is a report indicating that juvenile largemouth bass overwintering in
4°C have gills with thickened primary epithelia
(Leino, 1993). This may be
linked to reduced metabolic needs of the fish, which is torpid at this
temperature. Thus, it is possible that several fish species utilise
morphological alterations of the respiratory surface area as a strategy to
minimise ion loss and water influx at times of low oxygen needs. It could also
be mentioned that hypoxia may influence gill morphology in developing fish
larvae (McDonald and McMahon,
1977
).
Osmoregulation
We hypothesised that embedded lamellae might be beneficial to the
osmoregulatory ability of the fish. Plasma [Cl-] of saline-exposed
fish with embedded lamellae was therefore compared with that in fish with
protruding lamellae (Table 3).
The groups already differed in the control blood samples (before exposure to
hypersaline water), since [Cl-] of the blood plasma was
significantly lower in the group with protruding lamellae compared with the
group with embedded lamellae (Table
3; P<0.01). This indicates that when the crucian carp
develop protruding lamellae they do not fully compensate for the presumably
increased water influx and/or ion loss over the enlarged gill surface. After
1.5 h of hypersaline exposure, there was a significant increase in the blood
plasma Cl- content of the group with protruding lamellae
(P<0.01), while the Cl- content of the embedded
lamellae group was unaltered (Table
3; P>0.05). Exposure to saline water for 6 h increased
the Cl- blood plasma content significantly in both groups, but the
increase was significantly higher in the group with protruding lamellae
(Table 3; P<0.01).
All these data suggest that an increased respiratory surface area has a
significant effect on water and ion fluxes and probably increases the cost of
the ion pumping needed to maintain homeostasis.
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Cell proliferation and apoptosis
We next proceeded to investigate the cellular mechanisms underlying the
morphological changes. The total number of ILCM cells in each interlamellar
space, counted in the mid one-third of the filaments from the 2nd gill arch,
fell from 50±2 to 26±1 after 7 days of hypoxia
(Fig. 4; P<0.001).
From 7 to 14 days of hypoxia, the number of cells did not decrease
significantly (P>0.05), confirming the chronicle of change seen in
the SEM micrographs (Fig.
3D,E).
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To further examine the dynamics of the ILCM, we investigated the mitotic
activity by injecting the fish with BrdU, a synthetic analogue of thymidine,
and subsequently detecting cells in the S phase with an antibody against BrdU
(Alfei et al., 1993;
Tsai and Hwang, 1998
;
Uchida and Kaneko, 1996
).
Under normoxic conditions or in fish exposed to hypoxia for 3 or 7 days,
BrdU-labelled cells were rarely found in the lamellar epithelia, suggesting
that hypoxia has little effect on cell proliferation in the lamellae per
se. By contrast, ILCM cells showed mitotic activity under all conditions
(Fig. 4A-C). In fish kept in
normoxic water, S-phase cells made up 4.82±0.15% of the total cell
number in the ILCM (Fig. 5).
This fraction decreased significantly after 3 days (1.42±0.12%;
P<0.01) and 7 days (0.58±0.07%; P<0.01) of
hypoxia exposure, suggesting that reduced cell proliferation in the ILCM is
involved in reducing the size of this tissue and thereby causing the lamellae
to protrude after exposure to hypoxia.
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Next, we examined whether apoptosis could be involved in reducing the ILCM
during hypoxia. TUNEL was used to visualize apoptotic cells
(Gavrieli et al., 1992).
Apoptosis was rarely found in the lamellar epithelia, again suggesting that
this tissue was relatively stable. By contrast, the ILCM displayed a
significant apoptotic activity (Fig.
4D-F). Thus, under normoxic conditions, 0.78±0.09% of the
cells in the ILCM were TUNEL labelled (Fig.
5). After 1 day of exposure to hypoxia, the number of labelled
ILCM cells was already significantly increased (1.69±0.17%;
P<0.01). The apoptotic activity peaked after 3 days in hypoxia
(3.79±0.13%; P<0.01). Subsequently, the incidence of
apoptosis fell and, after 7 days of hypoxia, the degree of apoptosis was down
to pre-hypoxic levels (0.98±0.09%; P>0.05 compared with
normoxia). Thus, apoptosis was most frequent during the time when the ILCM was
undergoing a reduction in size, suggesting that apoptosis in the ILCM is
involved in reducing the size of this tissue, thereby causing the lamellae to
protrude after exposure to hypoxia.
Conclusions
We conclude that the morphological change occurring after exposure to
hypoxic conditions is caused by a combination of reduced cell proliferation
and an induction of apoptosis. To our knowledge, these results are the first
evidence of hypoxia-induced apoptosis in fish. Interestingly, S-phase and
apoptotic cells were spread throughout the ILCM
(Fig.4A-F), indicating that
factors such as type of cell or position in cell cycle, rather than
localisation, decide the fate of the ILCM cell. At present, we do not know if
this morphological hypoxia response occurs in all crucian carp populations. So
far, we have seen crucian carp with embedded lamellae in two populations some
500 km apart: Uppsala in Sweden and Oslo in Norway.
A likely evolutionary reason for having the capacity to alter the respiratory surface area is that it will make the fish able to minimise water and ion fluxes and maybe also reduce the risk of infections and uptake of toxic substances.
The ability of the ILCM cells to rapidly alter their apoptotic and mitotic
activity could make them interesting models for studying cellular mechanisms
involved in responses to different oxygen levels, particularly if they, like
other gill cells (Pärt et al.,
1993), can be cultured.
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Acknowledgments |
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