ORIGINAL PAPER
Immunohistochemical localization of constitutive and inducible Heat
Shock Protein 70 in carp (Cyprinus carpio) and trout (Oncorhynchus
mykiss) exposed to transport stress
C. Poltronieri, E. Negrato, D. Bertotto, D. Majolini, C. Simontacchi, G. Radaelli
Department of Experimental Veterinary Sciences, Faculty of Veterinary Medicine,
University of Padua, Italy
©2008 European Journal of Histochemistry
In the present work we investigated by immunohistochemistry the cellular localization of constitutive as well as
inducible heat shock protein 70 in several tissues of common carp (Cyprinus carpio) and rainbow trout
(Oncorhynchus mykiss) exposed to transport stress. In carp,
the constitutive form (HSC70) was detected only in red
skeletal muscle of both control and stressed animals. In the
same species, the inducible form (HSP70) was evident in
the epithelia of renal tubules, gills and skin of stressed animals, whereas in controls only red skeletal muscle exhibited
an immunopositivity to HSP70 antibody. In trout, immunostaining to HSC70 antibody was found mainly in the epithelia
of intestine, gills and skin of both control and stressed animals although the reactivity was generally higher in animals
exposed to transport stress. In the same species immunostaining to HSP70 antibody was observed only in red skeletal
muscle and epidermis of control animals.
Key words: HSP70, HSC70, immunohistochemistry,
transport stress, fish.
Correspondence: Giuseppe Radaelli,
Department of Experimental Veterinary Sciences
Viale dell’Università, 16/Agripolis,
35020, Legnaro (PD) Italy
Tel.: +39.049.790165.
Fax: +39.049.641174.
E-mail: giuseppe.radaelli@unipd.it
Paper accepted on July 24, 2008
European Journal of Histochemistry
2008; vol. 52 issue 3 (July-September): 191-198
F
ish are exposed to stressors in nature, as
well as in artificial conditions such as in
aquaculture, or in laboratories. These stressors, such as handling, weighing, crowding, grading, transportation, vaccination determine the
activation of the hypothalamic-pituitary-interrenal axis (HPI) and the release of corticosteroid
hormones such as cortisol (Wendelaar Bonga
1997, Mommsen et al. 1999). The generalized
stress response at the cellular level is characterized by a family of proteins referred to as the heat
shock proteins (HSPs) (Iwama et al. 1998).
Heat shock proteins (HSPs), also called stress
proteins, are a family of highly conserved cellular
proteins that are present in all cells in all life
forms (Morimoto et al. 1990, Iwama et al. 1998,
Feder and Hofmann 1999). Studies on different
model species have revealed three major families
of HSPs: HSP90 (85-90 kDa), HSP70 (68-73
kDa), and low molecular weight HSPs (16-24
kDa). In the unstressed cell, there is a constitutive
production of these proteins that are required in
various aspects of cellular homeostasis. HSP70 is
known to assist the folding of nascent polypeptide
chains, act as a molecular chaperone, and mediate
the repair and degradation of altered or denatured proteins. HSP90 is active in supporting various components of cell signaling, including the
cytoskeleton, enzymes, and steroid hormone
receptors. The low molecular weight HSPs have
diverse functions and it has been proposed that
they function as molecular chaperones, preventing
irreversible protein aggregation (Iwama et al.
2004). In fish, as in mammals, there are constitutive members (HSC70) of the heat shock proteins, which play important chaperoning role in
unstressed cells, and inducible (HSP70) forms,
which are expressed in detectable levels after
acute stressor insults (Iwama et al. 1998).
Heat shock protein 70 expression has been stud191
C. Poltronieri et al.
ied in fish after exposure to pesticides, virus, metals and other toxic compounds (Roy and
Bhattacharya 2006, Eder et al. 2007, Hansen et
al. 2007, Maradonna and Carnevali 2007).
Podrabsky and Somero (2007) found that an
inducible or embryo-specific form of HSP70 is
expressed during embryonic development in killifish (Austrofundulus limnaeus) and is elevated
during diapause II in this species. Gornati et al.
(2004, 2005) detected a different expression of
HSP70 and HSP90 mRNAs in sea bass farmed
at different stocking densities. Moreover, handling
and stocking density procedures can affect cortisol levels as suggested by several authors
(Marino et al. 2001, Skjervold et al. 2001,
Simontacchi et al. 2008). Recently, we demonstrated that inducible HSP70 mRNA, examined
by Real Time PCR, was significantly higher in skin
and skeletal muscle of sea bass exposed to transport stress than in controls (Poltronieri et al.
2007); in the same experimental conditions,
inducible HSP70 protein, detected by immunohistochemistry, was found only in skeletal muscle of
stressed animals (Poltronieri et al. 2007) suggesting that HSP70 mRNA and protein could be
potential candidates to describe welfare in fish.
The aim of this work was to examine the cellular localization of the constitutive and inducible
HSP70 proteins in two aquaculture species, common carp (Cyprinus carpio) and rainbow trout
(Oncorhynchus mykiss) before and after transport, one of the most stressful procedures in
aquaculture facilities. The localization of the two
proteins (HSC70 and HSP70) was investigated
by immunohistochemistry.
Materials and Methods
Animals
Adults of common carp (Cyprinus carpio) and
rainbow trout ( Oncorhynchus mykiss ) were
obtained from a freshwater fish-farm (Durigon,
Santa Cristina, Treviso, Italy). Some animals (7
carp and 10 trout) were sampled directly at the
farm immediately after capture (control animals). An equal number of fish from each species
was submitted to the same sampling procedure
after transport to the laboratory in aerated bags,
a journey of 1.5 hours for both species. For
192
immunohistochemistry, several tissues (intestine,
liver, pancreas, spleen, skeletal muscle, brain,
gills, kidney, skin) were removed and fixed in 4%
paraformaldehyde prepared in phosphatebuffered saline (PBS, 0.1 M, pH 7.4) at 4°C
overnight. All sampled animals were euthanased
using an overdose (300 mg/litre) of the anaesthetic MS-222, Sandoz, Italy.
Immunohistochemistry
Fixation and embedding
Samples fixed as described above were washed
in PBS, dehydrated through a graded series of
ethanol and embedded in paraffin. Sections were
cut at a thickness of 4 µm using a microtome.
Antisera
To localize the constitutive protein, a polyclonal anti-HSC70 antibody was used. The antibody
was raised in rabbit against a 13 residue synthetic peptide based on the human HSC70 (Stressgen
Biotechnologies, SPA-816, USA) and used at a
dilution of 1:2000. To localize the inducible protein, a monoclonal anti-HSP70 antibody was
used. The antibody was raised in mouse against a
purified HSP70 isolated from human Hela cells
(Stressgen Biotechnologies, SPA-810, USA) and
used at a dilution of 1:200.
Immunohistochemical procedure
Immunohistochemical staining was done using
the Envision system (goat anti-rabbit or goatanti-mouse immunoglobulins conjugated to peroxidase-labeled complex, Dako, Italy). Before
applying the primary antibody, endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 in methanol. Non-specific binding sites were blocked by incubating the sections
in 1:10 dilution of rabbit or mouse serum (Dako,
Italy). The immunoreactive sites were visualized
using diaminobenzidine (DAB) (Sigma, Italy) as
the chromogen. To ascertain structural details,
sections were counterstained with Mayer’s hematoxylin.
The specificity of the immunostaining was verified by incubating sections with: (1) PBS instead
of the specific primary antibodies (see above);
(2) normal rabbit serum instead of the primary
antibody; (3) PBS instead of the secondary antibody. The results of these controls were negative.
Original Paper
Results
In general, HSC70 antibody gave an immunostaining in numerous tissues of control as well as
stressed trout, whereas HSP70 antibody mainly
reacted with tissues from stressed carp (Table 1).
immunoreactivity to HSC70 antibody was also
observed in red skeletal muscle (Table 1).
Immunoreactivity to HSP70 antibody was
detected in red skeletal muscle and skin from
controls, whereas no immunostaining to inducible
HSP70 antibody was detected in all tissues from
stressed animals (Table 1).
Common carp
Immunoreactivity to HSC70 antibody was
detected only in red skeletal muscle which exhibited a moderate positivity in controls and a faint
immunostaining in stressed animals (Table 1). In
controls, a moderate immunoreactivity to HSP70
antibody was found only in red skeletal muscle,
whereas all other tissues were negative (Table 1;
Figure 1A, C, E). In stressed animals, HSP70
immunostaining was found in the apical cytoplasm and brush border of epithelial cells of renal
tubules (Figure 1B), in the epithelium lining primary lamellae of gills (Figure 1D) and in the epidermis of skin (Figure 1F).
Rainbow trout
Immunoreactivity to HSC70 antibody was
detected in both controls and stressed animals at
the level of: i) the epithelium of intestine (Figure
2A, B); ii) the epithelium lining both primary and
secondary lamellae of gills (Figure 2C, D) and iii)
the epithelium of skin (Figure 2E, F). In the
epithelia of intestine and gills from stressed animals (Table 1; Figure 2B, D) the immunoreactivity seems to be higher than that observed in controls (Table 1; Figure 2A, C). In controls, a faint
Discussion
In this study we detected the cellular localization of the constitutive (HSC70) and inducible
(HSP70) members of the heat shock protein 70
in adults of two aquaculture species, common
carp ( Cyprinus carpio ) and rainbow trout
(Oncorhynchus mykiss) exposed to transport
stress.
In fish the stress response has been categorized
into the primary, secondary and tertiary responses (Mazeaud et al. 1977, Wedemeyer et al.
1990). The primary response represents the perception of an alterated state which involves the
rapid release of stress hormones, catecholamines
and cortisol, into the circulation (Gamperl et al.
1994, Iwama et al. 1998). The secondary
response is defined as the manyfold immediate
actions and effects of stress hormones at blood
and tissue level, whereas tertiary response represents whole-animal and population-level changes
associated with stress (Wendelaar Bonga 1997,
Iwama et al. 1998). At the cellular level, fish
respond to stressors by an increased synthesis of
Table 1. Immunohistochemical localization of HSC70 and HSP70 in carp and trout exposed to transport stress.
Carp
Tissue
Skeletal
muscle
Brain
Intestinal epithelium
Kidney epithelium
Gill epithelium
Skin
Spleen
Liver
Pancreas
Trout
HSC70
HSP70
HSC70
HSP70
Controls
Stressed
Controls
Stressed
Controls
Stressed
Controls
Stressed
+R
-W
-
+/-R
-W
-
+R
-W
-
-R
-W
+
+
++
-
+/-R
-W\
+
*
++
++
-
-R
-W
++
*
+++
++
-
+/-R
-W
*
++
-
-R
-W
*
-
Staining: -, not detectable; +/-, slight but above background levels; + moderate; ++, marked staining; +++, strong. *Tissue not examined in this species. R, W: red and white muscle
fibres.
193
C. Poltronieri et al.
Figure 1. Immunohistochemical localization of inducible HSP70 in adults of carp. A, C, E: Control animals. B, D, F: stressed animals. All panels are counterstained with haematoxylin. A) The parenchyma of the kidney is negative. B) In stressed animals, epithelial cells of renal tubules
show immunostaining at the level of the apical cytoplasm and brush border (arrows). C) Epithelium of gill filaments is negative. D) In stressed
animals, a moderate immunostaining is present in the epithelial cells lining the secondary lamellae of the gills (arrows). E) Epidermis is negative. F) In stressed animals, epithelial cells of skin show a marked immunostaining. Bars: A, 20 µm; B, 10 µm; C-D, 12.5 µm; E-F, 20 µm.
194
Original Paper
Figure 2. Immunohistochemical localization of constitutive HSC70 in adults of trout. A, C, E: Control animals. B, D, F: stressed animals.
All panels are counterstained with haematoxylin. A) The epithelium of the intestine shows rare positive cells (arrows). B) In stressed animal, intestinal epithelium exhibits a marked immunoreactivity at the level of goblet cells (arrows). C-D) A marked immunostaining is present in the epithelial cells lining both the primary and secondary lamellae of the gills, although in stressed animals (D, asterisks) the reactivity is stronger than in controls (C, arrowheads). E-F) In skin of both control and stressed animals, epithelial cells exhibit a marked
immunostaining. Bars: A-B, 12.5 µm; C-D, 10 µm; E-F, 20 µm.
195
C. Poltronieri et al.
HSPs (Iwama et al. 1998) suggesting the use of
these proteins as indicators of stressed states in
fish. The aim of this work was to investigate the
use of HSP70 family as indicator of transport
stress in fish; this family includes several members of proteins, some of which are constitutively
(HSC70) expressed under normal growth conditions and others which are inducible (HSP70)
under various stress conditions (Morimoto et al.
1990). In our experimental condition the stress
response to transport has been evaluated by
measuring plasma cortisol levels demonstrating a
significantly increased of cortisol levels in
stressed animals (these results are object of
another paper already submitted for publication).
The expression of HSP70 mRNA has been studied in numerous fish species subjected to different
stress conditions (Sanders et al. 1995, Williams
et al. 1996, Vijayan et al. 1997, 1998, Schmidt
et al. 1998, Duffy et al. 1999, Hassanein et al.
1999, Currie et al. 2000, Ali et al. 2003a, b,
Zarate and Bradley 2003, Gornati et al. 2004,
Deane and Woo 2005, Ojima et al. 2005). Our
group demonstrated by Real-Time PCR an
increased expression of inducible HSP70 mRNA
in 40 day larvae and 80 day fry of sea bass
exposed to transport stress (Poltronieri et al.
2007); moreover, muscle and skin of stressed
adults showed an higher significant expression of
inducible HSP70 mRNA respect than controls
(Poltronieri et al., 2007). In the same species,
Gornati et al. (2004) detected an increased
expression of HSP70 after overcrowding exposure.
Since most inducible HSPs do not contain
introns, their mRNA is quickly translated into
protein within few minutes of exposure to stressors (Mayer et al., 2000). For this reason, in the
present work expression of HSP70 has been
examined by immunohistochemistry in order to
detect the cellular localization of both constitutive and inducible proteins after a transport
stress.
Technical specifications from the company indicate that the two commercial antibodies used for
this immunohistochemical investigation distinguish between the constitutive and inducible
forms of HSP70; above all the inducible HSP70
antibody detects a protein, corresponding to the
apparent molecular mass of inducible HSP70 on
SDS-PAGE immunoblots, in samples from differ196
ent vertebrate species, including carp.
In this work immunopositivity to HSC70 antibody has been found only in red skeletal muscle of
both control and stressed carp, although the constitutive member of HSP70 is required in the
unstressed cells in various aspects of metabolism
to maintain cellular homeostasis (Fink and Goto
1998). These findings are in contrast to the ubiquitous distribution of HSC70 protein found by
immunohistochemistry during ontogenesis of controls as well as transported sea bass (Poltronieri
et al. 2007). Moreover, Ali et al. (2003a, b)
observed a tissue- and stressor-specific differential expression of two HSC70 genes in carp. It
would be interesting to examine the expression of
HSC70 by Real-Time PCR in order to verify a
possible effect of transport stress on mRNA
expression of carp. Interestingly no immunoreactivity for inducible HSP70 protein has been
observed in most of tissues from control carp;
only red skeletal muscle exhibited a moderate
immunostaining. Barton and Iwama (1991) suggested that the presence of HSP70 protein in red
skeletal muscle is in agreement with an increased
muscular activity. A novel observation in the present study is the immunolocalization of inducible
HSP70 protein in the epithelia of renal tubule,
gills and skin of stressed carp. In the same tissues
from controls the immunostaining was negative
suggesting that inducible HSP70 is required for
allowing cells to cope with stressor events (Boone
and Vijayan 2002). Moreover, the immunopositivity observed in skin epithelium agrees with the
expression of inducible HSP70 mRNA observed
in sea bass after transport stress (Poltronieri et
al. 2007).
In trout, the pattern of immunoreactivity for
constitutive HSC70 was similar to that observed
in sea bass (Poltronieri et al. 2007) since several
tissues from both control and stressed animals
exhibited a positivity. In the same species,
Zafarullah et al. (1992) observed that HSP70
mRNA is constitutively expressed in different
trout tissues. Moreover, in our experiments, in the
epithelia of intestine and gills from stressed animals the immunostaining was higher than in controls suggesting an increased expression of constitutive HSC70 after transport. In the present
study inducible HSP70 protein has been detected
in red skeletal muscle and in skin epithelium from
control trout whereas no immunoreactivity was
Original Paper
found in all tissues from stressed animals. Lack
of immunoreactivity in all tissues from stressed
trout is in contrast to the expression of inducible
HSP70 protein observed in the epithelia of renal
tubules, gills and skin from stressed carp suggesting that in trout the mRNA has not been translated into a protein. Moreover, our results are in
agreement with those observed by Washburn et
al. (2002) who suggested that the capture and
transport of trout for environmental monitoring
purposes should not interfere with the use of
stress proteins as biomarkers.
In conclusion, the different cellular distribution
of both constitutive and inducible HSP70 proteins suggest that transport stress can affect
their expression.
Acknowledgements
This research was supported by a grant of
Ministero dell'Università e della Ricerca
Scientifica e Tecnologica (ex 60%). The authors
wish to thank the team of the fish farm for providing fish. The authors wish to thank Mr.
Giovanni Caporale for skillful technical work.
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