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Aquatic Toxicology 86 (2008) 131–141
Development of a marine fish model for studying in vivo
molecular responses in ecotoxicology
R.Y.C. Kong a , J.P. Giesy a,b , R.S.S. Wu a , E.X.H. Chen a , M.W.L. Chiang a ,
P.L. Lim c , B.B.H. Yuen a , B.W.P. Yip a , H.O.L. Mok a , D.W.T. Au a,∗
a
b
Centre for Coastal Pollution and Conservation, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong
Department of Veterinary Biomedical Sciences & Toxicology Center, University of Saskatchewan, Saskatoon, SK, Canada
c Clinical Immunology Unit, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong
Received 15 August 2007; received in revised form 16 October 2007; accepted 19 October 2007
Abstract
A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization
(ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections
(5 ␮m) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins
in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, Vv ) was used to quantify
ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen
(PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish
system. Using chronic hypoxia (1.8 ± 0.2 mg O2 L−1 for 3 months) as an environmental stressor, we were able to identify significant alterations
in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma
(p < 0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to
stresses in the marine environment.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Hypoxic stress; Histology; Immunohistochemistry; ISH; Telomerase
1. Introduction
A variety of molecular, biochemical and histological
responses in fish have been employed as biomarkers of various environmental stresses (Gavilán et al., 2001; Au, 2004;
Facey et al., 2005; Hutchinson et al., 2006). While these suborganismal responses are sensitive, reproducible and easier to
determine, most of them are tissue- and/or cell-specific. For
instance, CYP1A1 mRNA expression and EROD enzyme induction are different in liver (hepatocytes), intestine (enterocytes)
and gill (epithelia) of fish, depending on the exposure route
of toxicants (Wong et al., 2001; Yuen and Au, 2006). Current
molecular techniques such as real-time PCR and Western blotting and chemical analysis of metabolites normally only provide
information for individual tissues or the whole animal, but are
∗
Corresponding author. Tel.: +852 2788 971; fax: +852 2788 7406.
E-mail address: bhdwtau@cityu.edu.hk (D.W.T. Au).
0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2007.10.011
unable to differentiate molecular changes among different cell
types within the tissue.
Recently, there is a trend of using small size fish as sentinel
vertebrate species for ecotoxicology and biomedical research
(Moore, 2002; Wittbrodt et al., 2002; Hawkins et al., 2003;
Hinton et al., 2005). Small fish have several advantages in ecotoxicological studies, since they are generally easy to maintain
and breed under laboratory conditions. The generation time is
relatively short, and fish can produce eggs regularly, hence providing a variety of developmental and reproductive endpoints
for whole life cycle and multi-generation assessments. To this
end, the zebrafish (Danio rerio), fathead minnow (Pimephales
promelas), mosquito fish (Gambusia affinis), guppy (Poecilia
reticulata) and Japanese medaka (Oryzias latipes) have been
commonly used as freshwater fish models in ecotoxicological
studies (Dodd et al., 2000; Castro et al., 2004; Wolf et al., 2004;
Volz et al., 2005; Carter and Wilson, 2006; Kissling et al., 2006).
Surprisingly, a fish model for assessing environmental stress
in the marine environment has not been developed, although
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a number of freshwater/estuarine species (e.g. the sheepshead
minnow Cyprinodon variegatus and the mummichog Fundulus heteroclitus) has also been commonly adapted to seawater
for toxicological studies due to their hardiness in captivity.
However, both C. variegatus and F. heteroclitus suffer from
varying rates of growth, which compromises equilibrating toxic
responses, and poor knowledge of their genomes make molecular toxicological studies on these species difficult. Further, the
mummichog takes 2 years to reach sexual maturity, and this
makes it difficult to carry out whole life cycle studies.
The marine medaka Oryzias melastigma (McClelland) has
a number of attributes that render it a potentially good marine
fish for ecotoxicological studies. O. melastigma is small and
easy to culture and breed, and it completes the whole life
cycle in seawater. Marine medaka are similar to freshwater medaka O. latipes, they exhibit uniform growth, which
confer an additional advantage in using this species for ecotoxicological studies. Phylogenetically, this species is closely
related to O. latipes, of which the entire genome has been
worked out recently (Kasahara et al., 2007). The anatomy, biology and nutritional requirements of O. melastigma is similar
to that of O. latipes, which are well known and an atlas is
available (Anken and Bourrat, 1998; Fujita et al., 2006). In
addition, much of the information on the physiology of O.
latipes (http://www.bio.nagoya-u.ac.jp:8000/Welcome.html) is
also applicable for O. melastigma.
Notwithstanding, the use of small fishes for tissue-specific
molecular analyses presents a major challenge. The quantity
of a specific tissue available for analysis is often very limited,
and isolation of such a small amount of tissue is often difficult
and time consuming. However, this limitation can be overcome
by using in situ hybridization (ISH) and immunohistochemistry
(IHC) analyses on preserved whole fish tissues. With the recent
advent of image analysis software, the effectiveness of stereological analysis for quantification of IHC and ISH signals on tissue
sections has been greatly enhanced. Moreover, various technical problems are associated with the fixation and sectioning of
relatively large-sized adult fish specimens (ca. 3 mm) (Weber et
al., 2002), due mainly to its heavy bony structures. Traditional
decalcification of bony structures using formic acid or EDTA not
only lead to poor RNA preservation, but are also time consuming
(and may require up to 7 days) (Moore et al., 2002). Previous
fixation protocols for adult small fish, e.g. the Japanese medaka,
zebrafish, guppy and mosquito fish were specifically designed
for histopathologic evaluation (Fournie et al., 1996; Neely et al.,
2002; Zodrow et al., 2004) and a few on immuno-localization
studies (Ortego et al., 1994; Moore et al., 2002; Ko et al., 2005).
Until now, no protocols have been developed for the parallel
detection of mRNA and protein molecules in whole tissues of
these adult small fish. In the first part of this study, we have
developed and optimized procedures for the fixation and processing of whole adult marine medaka, enabling the production
of tissue sections suitable both for subsequent ISH and IHC
analyses.
Telomerase is an enzyme involved in cell immortalization,
carcinogenesis and tissue regeneration (Greider, 1998). The catalytic subunit telomerase reverse transcriptase (TERT) has been
shown to regulate cell proliferation, mediate apoptosis, promote
DNA repair and cell survival in vitro (see review of Chung et al.,
2005), which suggest that TERT has a central role in controlling
in vivo cell growth and tissue homeostasis. In fish, expression
of TERT gene has been found in a variety of somatic tissues
in O. melastigma (Yu et al., 2006) and Fugu rubripes (Yap et
al., 2005). In the second part of this study, we employed the
omTERT mRNA and protein and proliferating cell nuclear antigen (marker for cell proliferation) as molecular endpoints to
demonstrate the feasibility of using ISH and IHC techniques to
simultaneously localize and measure in vivo expression levels
of these gene and proteins in different tissues of a single medaka
fish, and to allow statistical analysis to be made on the correlation
of these gene and protein expression data in a whole fish.
Hypoxia has now become a pressing environmental problem
in aquatic systems worldwide (Diaz, 2001; Wu, 2002). Hypoxia
has been reported to up-regulate TERT expression in liver of
O. melastigma (through the hypoxia-inducible factor-1, HIF-1)
(Yu et al., 2006), which may perturb normal cell proliferation
and apoptosis in hepatocytes. In the third part of this report, we
used hypoxia as a model stressor and the liver as a model organ
to study the stress responses of omTERT mRNA (by ISH) and
protein (by IHC), cell proliferation (by PCNA) and apoptosis
(by the terminal dideoxynucleotidyl-mediated dUTP nick end
labeling, TUNEL) in O. melastigma. The overall objective of
this study is to demonstrate that O. melastigma can serve as a
good marine fish model for ecotoxicology, and enable in vivo
molecular responses at the nucleic acid and protein levels to be
localized and measured simultaneously in different tissues/cells
of the same individual.
2. Materials and methods
2.1. Marine medaka
Marine medaka were purchased from a commercial hatchery in Taiwan and were maintained in 30‰ artificial seawater
at 5.8 ± 0.2 mg O2 L−1 , 28 ± 2 ◦ C in a 14-h light:10-h dark
cycle. Under optimal growth and breeding conditions, adult
O. melastigma produced sufficient numbers of genetically
homogeneous offspring. For the chronic hypoxia exposure
experiment, 4-week old O. melastigma were used and divided
into two groups. The first group was maintained in a hypoxic
system (1.8 ± 0.2 mg O2 L−1 ) and the second group under normoxia (6.4 ± 0.2 mg O2 L−1 ). Dissolved oxygen was monitored
continuously using DO meters and polarographic probes (ColeParmer 5643-00, IL, USA). A detail design of the hypoxic and
normoxic systems is given in Shang et al. (2006). At the end of a
3-month exposure period, whole adult O. melastigma were fixed
and processed for parallel analyses by ISH (omTERT gene), IHC
(TERT protein and PCNA) and TUNEL (apoptosis).
2.2. Fixation and processing of whole adult O. melastigma
Adult O. melastigma (ca. 3.5 in cm length) were immobilized in ice-chilled water. Fixative was flushed over the gills and
injected into the abdominal cavity through the mouth using a
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133
Table 1
Summary of fixation and processing regimes for histological and molecular analyses of whole adult O. melastigma
Trials
Fixation (overnight at 4 ◦ C)
Clearing medium
Processing
temperature
Evaluation
Sectioning
quality
Morphology
Labeling efficiency
IHC
ISH
1
4% PFA in 0.1 M CB
4% PFA in PBS
1:1 8% PFA:NSW
Xylene
Xylene
Xylene
RT
RT
RT
Poor
Poor
Poor
Good
Moderate
Poor
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2
HC
HC with 1% sucrose
HC with 5% sucrose
HC with 10% sucrose
Xylene
Xylene
Xylene
Xylene
RT
RT
RT
RT
Good
Good
Good
Good
Poor
Moderate
Moderate
Poor
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4% PFA in 0.1 M CB followed by HC with
1% sucrose
2% PFA & 2.5% Glut in 0.1 M CB followed
by HC with 1% sucrose
4% PFA in 80% HC with 1% sucrose
Chloroform
RT
Moderate
Good
n.d.
n.d.
Chloroform
RT
Moderate
Best
n.d.
n.d.
Chloroform
RT
Good
Good
n.d.
n.d.
Chloroform
4 ◦C
Best
Best
n.d.
n.d.
Chloroform
4 ◦C
Best
Best
Good
n.d.
Chloroform
4 ◦C
Best
Best
Best
Good
Chloroform
4 ◦C
Best
Best
Best
Best
3
4
5
2% PFA and 0.2% Glut in 80% HC with 1%
sucrose
2% PFA and 0.05% Glut in 80% HC with
1% sucrose
2% PFA and 0.05% Glut in 80% HC with
1% sucrose and 1% CaCl2
2% PFA and 0.05% Glut in 80% HC with
1% sucrose and 1% CaCl2 followed by
infiltration in gum sucrose
PFA, Paraformaldehyde; CB, cacodylate buffer; PBS, phosphate-buffered saline; NSW, natural seawater; HC, HistoChoice; Glut, glutaraldehyde; n.d., not determined.
small syringe. A small hole was punched into the swim bladder
through the mid-dorsal body wall with a fine pin, and the body
was gently pressed to release the gas. The body was slit open
along the mid-ventral line (from the anal vent to the operculum) to facilitate rapid penetration of the fixative. Hard tissues
(including skull roof, otoliths, operculum and fins) were gently removed and fish were immersed in fixatives overnight at
4 ◦ C (Table 1). Fixed fish were dehydrated in a 70/80/95/100%
graded series of methanol (20 min × 2) followed by clearing in
chloroform (30 min × 3 and 15 min × 2) at 4 ◦ C. All processing
steps (from fixation to clearing) were conducted in a tissue rotator (FINEPCR, Korea) at 10 rpm. Dehydrated O. melastigma
were infiltrated for 2 h (30 min × 4) at 55 ◦ C in melted paraffin
(Paraplast X-TRA from Tyco Healthcare, UK) before embedding. Fish were orientated diagonally inside an embedding mold
(EMS, USA) with the head pointing toward the bottom lefthand corner of the mold. Solidified blocks were stored at 4 ◦ C
until ready to use. Serial sagittal sections (5 ␮m) were cut on a
rotary microtome (Leica RM2125, Germany) and mounted onto
Superfrost® Plus slides (Menzel-Gläser, Germany). All sections
collected from a single fish were number-coded for subsequent
ISH and IHC experiments. Tissue sections were dried overnight
on a slide warmer at 37 ◦ C before analyses.
2.3. Whole fish in situ hybridization
A 300-bp omTERT fragment spanning motif E and motif E-I
(nt. 2749–3048; Yu et al., 2006) was cloned into the pGEM-
T Easy vector (Promega, Madison, USA), and digoxigenin
(DIG)-labeled antisense and sense riboprobes were synthesized by in vitro transcription. Deparaffinized and rehydrated
O. melastigma tissue sections were digested with proteinase
K (1 mg mL−1 ) at room temperature for 20 min and post-fixed
in 4% paraformaldehyde in PBS for 20 min. Hybridization
was performed overnight at 50 ◦ C with DIG-labeled riboprobes
(0.5 ng ␮L−1 ) in 2× SSC, 50% formamide, 10% dextran sulfate,
50 ␮g mL−1 yeast tRNA and 5 U mL−1 RNase inhibitor. After
hybridization, slides were washed in a graded series of SSC,
0.1% Tween 20 at room temperature and incubated with antiDIG antibody coupled to alkaline phosphatase (at 1:100 dilution;
Roche Applied Science, Germany). Signals were detected using
the NBT/BCIP substrate (Zymed, South San Francisco, USA).
Sections were counterstained with Nuclear Fast Red and then
examined by light microscopy (Axioplan 2 imaging; Carl Zeiss,
Germany).
2.4. Western blotting
Cytoplasmic and nuclear proteins were extracted from adult
fish organs (gill, liver, kidney and intestine, testes, ovary,
brain and muscle) using NE-PER® Nuclear and Cytoplasmic
Extraction Reagents (Pierce Biotechnology, Rockford, USA)
according to the manufacturer’s instructions. Each organ from
5 to 10 fish was pooled for protein extraction. Cytoplasmic
and nuclear proteins (10–40 ␮g) were separated in 7% SDS
PAGE, transferred to PVDF membranes (GE Healthcare Bio-
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Sciences, NJ, USA), blocked in 5% non-fat dry milk (Nestle,
Shuangcheng, People’s Republic of China) in 0.1% TPBS (0.1%
Tween 20 in 0.1 M Na2 HPO4 , 0.1 M NaH2 PO4 , 0.5 M NaCl,
pH 7.2) and incubated overnight at 4 ◦ C with mouse antihuman TERT monoclonal antibody mAb476 (1:1000) (Leung
et al., 2005). Non-specific binding of antibody was washed off
with three changes of 0.1% TPBS followed by detection with
HRP-conjugated secondary anti-mouse IgG (1:5000 dilution in
5% non-fat milk in 0.1% TPBS) at room temperature for 1 h.
After washing with three rounds of 0.1% TPBS, the signal was
detected with ECL Plus Western Blotting Detection System (GC
Healthcare) and the membranes were exposed to HyperfilmTM
ECLTM (GE Healthcare).
2.5. Immunohistochemistry (omTERT and PCNA)
Deparaffinized and rehydrated O. melastigma tissue sections were incubated for 15 min in 100% methanol containing
0.03% H2 O2 to quench endogenous peroxidases. For immunolocalization of omTERT protein, antigen retrieval in Enhancing
buffer (IDL Biotech, Bromma, Sweden) for 20 min was followed by incubation with anti-hTERT monoclonal antibody
mAb476 (1:100 diluted) (Leung et al., 2005) at room temperature for 2 h. For PCNA staining, after heat retrieval by
microwave in citrate buffer (pH 6.0) for 15 min, fish sections
were incubated with monoclonal antibody (no. M0879) for
PCNA antigen (1:5000; DakoCytomation, Denmark) at room
temperature for 1 h. For both omTERT and PCNA staining, treatment with the primary antibodies were followed by incubation
with HRP-conjugated secondary goat anti-mouse IgG antibody (1:5000 dilution in 1% BSA in PBS; Dako EnvisionTM +
System, Denmark) at room temperature for 30 min. Sections
were rinsed in PBS and signals detected with the chromogenic
substrate, 3,3 -diaminobenzidine chromogen (DAB) (DakoCytomation, Denmark). Sections were counterstained with Mayer’s
Hematoxylin (DakoCytomation, Denmark) and mounted using
Permount (Fisher Scientific). Negative controls were prepared
by substitution of the primary antibody with non-immune
sera.
2.6. TUNEL assay
Apoptotic cell death was determined using terminal
dideoxynucleotidyl-mediated dUTP nick end labeling (TUNEL)
according to the manufacturer’s instructions (Roche Applied
Science, Germany). Tissue sections on slides were rinsed with
PBS, incubated in permeabilization solution (0.1% Triton X100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with
PBS, incubated with 10 ␮l TUNEL reaction mixture (terminal
deoxynucleotidyl transferase 200 U mL−1 , FITC-labeled dUTP
10 mM, 25 mM Tris–HCl, 200 mM sodium cacodylate, 5 mM
cobalt chloride) for 60 min at 37 ◦ C, and then rinsed 3× with
PBS. For visualization of TUNEL signals, tissue sections were
incubated with 10 ␮l Converter-POD (anti-FITC antibody, Fab
fragment from sheep, conjugated with horseradish peroxidase)
for 30 min, rinsed 3× with PBS, and then stained with DABsubstrate solution (DakoCytomation, Denmark) for 10 min in
the dark at room temperature. Negative control was obtained
by omitting TdT from the TUNEL reaction mixture. Sections
were counterstained with Mayer’s hematoxylin (DakoCytomation, Denmark) for 2 min and a cover slip was mounted with
a drop of pure Permount® (Fisher Chemical, USA). Only the
brown and condensed nuclei with fragmented nucleoli showing the characteristic apoptotic morphology were counted as
apoptotic nucleus.
2.7. Stereological analysis
Expression of omTERT mRNA and protein, PCNA and
TUNEL signals were measured by volume density (Vv ), according to the stereological principles of Weibel (1979) and Reed and
Howard (1998). Vv represents the volume fraction of positively
stained cells in the fish target tissue and was calculated according
to Eq. (1):
Vv =
Pi
PT
(1)
where Pi is the number of test points enclosed within the profiles
of the particulate structure investigated (i.e., omTERT mRNA
Table 2
Stereological quantification of omTERT, PCNA and TUNEL positive signals in tissues of adult O. melastigma detected by ISH and IHC
Organ
Fish replicates
Magnification
Test system, d (␮m)
Number of test
points
Number of fields
for analysis
Stereological parameter: volume
density, Vv (Pi , reference space)
Gonad
Testis
5 males
200×
Square lattice, d = 20
192 (16 × 12)
Vv (positive signals, sperm cysts)
5 females
200×
Square lattice, d = 20
192 (16 × 12)
Kidney
Intestine
5 males/5 females
5 males/5 females
600×
600×
Square lattice, d = 5
Square lattice, d = 5
432 (24 × 18)
432 (24 × 18)
Whole organ on
section
Whole organ on
section
15
10
Gill
5 males/5 females
600×
Square lattice, d = 5
432 (24 × 18)
15
Liver
5 males/5 females
600×
Square lattice, d = 5
432 (24 × 18)
10
Muscle
5 males/5 females
600×
Square lattice, d = 5
432 (24 × 18)
30
Ovary
Vv (positive signals, ovarian follicles)
Vv (positive stained nuclei, kidney)
Vv (positive stained nuclei, intestinal
epithelia)
Vv (positive stained nuclei, gill
filament and lamella)
Vv (positive stained nuclei, liver
parenchyma)
Vv (positive stained nuclei, muscle
fibres)
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135
Fig. 1. omTERT immunostaining patterns in sagittal sections of whole adult (16-week old) marine medaka, O. melastigma. Immunohistochemistry was performed
using anti-human TERT monoclonal antibody on male (A) and female (B) fish sections, and signal detection was accomplished with diaminobenzidine (DAB).
(Sections were counterstained with hematoxylin.) Br, Brain; Bb, backbone; E, eye; G, gill; Gb, gall bladder; Gu, gut; H, heart; K, kidney; L, liver; M, muscle; O,
ovary; Sb, swimming bladder; Sc, spinal cord; T, testis. Scale bar = 2 mm. (Insets) External morphologies of fish of the respective sex.
and protein, PCNA or TUNEL-positive signals) and PT is the
number of test points falling on the reference space (i.e., the fish
tissue being investigated). Stereological analysis was performed
on each fish tissue with the aid of the Computer Assisted Stereology Toolbox (CAST) 2.0 software (Olympus, Denmark). Images
were captured using the Olympus BX51 compound microscope
(Denmark) equipped with a motorized stage (ProScan; PRIOR
Scientific, USA) and a 3-CCD color video camera (KY-F58;
JVC, Japan). The stereological procedures for quantitative ISH
and IHC analyses are summarized in Table 2.
2.8. Statistical analyses
Pearson product-moment correlation analysis was used to
relate the in vivo expression levels of omTERT mRNA and
protein, and their relationships to PCNA in gonad, kidney, intestine, gill, liver and muscle of the same fish (n = 5 for each
sex). Student’s t-test was used to test the null hypothesis that
there were no quantitative changes in particulate expression levels (Vv of omTERT mRNA-, omTERT protein-, PCNA- and
TUNEL-positive stained nuclei) between the normoxic control
and hypoxic samples. Significance level (α) was set at 0.05 (Zar,
1996).
3. Results
3.1. Whole adult O. melastigma fixation and processing
The protocol that yielded high quality tissue sections with
optimal preservation of tissue and cellular morphology, nucleic
acid and antigenicity was developed by optimizing various fixation and processing regimes that included testing the efficacy
of: (i) fixative combinations; (ii) clearing media and (iii) processing temperatures (Table 1). Among all of the conditions
tested, primary fixation of adult O. melastigma in a cocktail
of 0.05% glutaraldehyde (Glut), 2% paraformaldehyde (PFA)
Fig. 2. Subcellular localization of omTERT protein in different tissues of adult O. melastigma. Western blot analysis of omTERT was performed on cytoplasmic
(Cy) and nuclear (Nu) fractions from representative reproductive (A), somatic (B) and regenerative (C) tissues of adult O. melastigma (4-month old).
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Fig. 3. Parallel in situ hybridization and immunohistochemical staining patterns for omTERT mRNA, omTERT protein and PCNA in reproductive (testis and
ovary) (A–F), somatic (brain and muscle) (G–L) and regenerative (gill, intestine, kidney and liver) (M–X) tissues of O. melastigma. Serial sections in the left
column were hybridized with omTERT antisense riboprobe and immunodetected with anti-DIG antibody coupled to alkaline phosphatase. Signals were visualized
using NBT/BCIP as substrate (purple, positive signal). Sections were counterstained with nuclear fast red (red, negative signal). Sections in the middle and right columns
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and 1% sucrose and 1% CaCl2 in Amresco® HistoChoiceTM
MB for 16 h at 4 ◦ C, followed by immersion in Holt’s gum
sucrose solution (1% aqueous gum arabic [Sigma, St. Louis,
USA] and 30% aqueous sucrose) (Holt et al., 1960) at 4 ◦ C for
24 h yielded the best results in section quality and preservation
of tissue morphology, nuclei acid and antigenicity (Table 1).
Because xylene rendered fish tissues too brittle for sectioning,
chloroform was used instead as the clearing agent. Likewise,
PFA fixation is generally favorable for the preservation of tissue and cellular morphology but poor for sectioning owing
to tissue brittleness, and hence HistoChoice (HC) was used
to soften bony tissues, rendering the fixed adult fish easier to
section, and no decalcification step (in formic acid, EDTA)
was required. Fixation of whole adult fish in a cocktail of HC
with 1% sucrose in 2% PFA and 0.05% Glut at 4 ◦ C considerably improved tissue preservation and sectioning quality
without weakening IHC labeling efficiency. Fatty tissues were
better preserved by low-temperature processing. Infiltration of
fixed whole fish tissues in gum-sucrose overnight at 4 ◦ C further enhanced tissue preservation as well as efficiency of ISH
and IHC labeling. The quality of tissue preservation using this
regime was equally satisfactory for both male (Fig. 1A) and
female fish (Fig. 1B). By and large, at least 200 serial sagittal
whole fish sections could be obtained from a single fish allowing multiple fish organs/tissues to be examined simultaneously
on a single section for parallel histological evaluation and quantitative mRNA/protein localization after ISH/IHC, respectively
(Fig. 1A and B).
3.2. Western blotting
Specific hybridization of the mouse anti-human TERT
monoclonal antibody (mAb476) to the medaka omTERT protein (expected size of 120 kDa) was detected in nuclear
extracts from gill, liver, testes, brain and muscle tissues of
O. melastigma (Fig. 2). However, no protein band of 120 kDa
in size was detected in extracts from fish kidney, intestine
and ovary. Sequence alignment of the hTERT protein at aa
543–668 (region recognized by mAB476) with the corresponding region in the omTERT protein (aa 507–621) showed
high sequence similarity (53%) between the human and fish
sequence.
3.3. Tissue-specific expression revealed by ISH and IHC on
whole fish sections
ISH using DIG-labeled antisense riboprobes revealed distinct localization patterns of omTERT mRNA in various
137
organs of adult O. melastigma. No hybridization signal
was observed in fish tissues with the sense omTERT riboprobe (negative control). Similarly, immunoreactivity of the
mAb476 and M0879 antibodies for the omTERT protein
and PCNA, respectively, are cell-type specific although differences in subcellular distribution patterns were observed
for the two proteins (Fig. 3A–X). No immunoreactivity was
observed in fish tissues with non-immune sera in the negative
controls.
In testis, high expression of omTERT mRNA (ISH), and
strong omTERT protein and PCNA immunoreactivity were
detected in dividing spermatogonia (Sg), spermatocytes (Sc)
and spermatids (St), but not in differentiated spermatozoa (Sz)
(Fig. 3A–C). In ovary, strong expression of omTERT mRNA
was detected in the cytoplasm of pre-vitellogenic (PV) oocytes
(Fig. 3D–F), while omTERT protein and PCNA were localized
in the nuclei of PV oocytes and granulosa cells of vitellogenic oocytes. In brain, while both omTERT mRNA and
omTERT protein were detected in the granule cell mass of
telencephalon, optic tectum and cerebellum (Fig. 3G–I), with
intense omTERT immunostaining being observed in the cytoplasm; PCNA-positive cells were clearly evident at the borders
between the telencephalon and diencephalon, and between the
mesencephalon and rhombencephalon, which are the proliferative zones of the brain. In muscle, omTERT mRNA was
only weakly expressed (Fig. 3J), with omTERT protein and
PCNA immunostaining being detected only in the nuclei of
fibroblasts and muscle fibers (Fig. 3K and L). The relative abundance of PCNA-positive cells was less than those that showed
positive immunoreactivity for the omTERT protein. In gill,
PCNA-positive cells and cells expressing omTERT mRNA were
localized along the epithelial filaments of the gill (Fig. 3M–O),
while cells showing omTERT immunoreactivity were evident
in the epithelia of both gill filaments and lamellae. In intestine, expression of omTERT mRNA and protein were detectable
in enterocytes of the mucosal epithelia (Fig. 3P–R), whereas
PCNA staining was confined to the nuclei of proliferative enterocytes near the crypt region of the mucosal folds. In kidney,
omTERT mRNA was only modestly expressed (Fig. 3S–U) while
omTERT immunostaining was most prominent in renal epithelial cells (both nucleus and cytoplasm) and in the adjoining
lymphoid tissues. PCNA immunostaining was strong in lymphoid tissues but sporadic in renal epithelial cells. In liver,
omTERT mRNA was moderately expressed across the liver
parenchyma except the red blood cells (Fig. 3V–X). omTERT
and PCNA immunostaining were confined to the nucleus of
hepatocytes.
are consecutive tissue sections of those on the left, and were immunostained with anti-hTERT (mAb476) and anti-PCNA (M0879; DakoCytomation) monoclonal
antibodies, respectively. Signal detection was accomplished with DAB (brown, positive signal). Sections were counterstained with hematoxylin (blue, negative
signal). Testis (A–C): Sg, spermatogonia; Sc, spermatocyte; St, spermatid; Sz, spermatozoa. Ovary (D–F): PV, previtellogenic oocyte; cy, cytoplasm; n, nucleus; pn,
provitelline nucleoli; VP, vitellogenic oocyte; yv, yolk vacuole; gc, granulose cell; af, attaching filament; tc, theca cell. Brain (G–I): Tel, telencephalon; OT, optic
tectum; Ce, cerebellum; Rh, rhombencephalon; Hyp, hypothalamus. Skeletal muscle (J–L): MF, muscle fiber; n, nucleus. Gill (M-O): La, lamella; F, filament; CC,
chloride cell; PC, pillar cell; MC, mucus cell; EC, epithelial cell; Ery, erythrocyte. Intestine (P–R): Lp, lamina propria; Me, mucosal epithelium; M, muscularis; Sm,
serous membrane. Kidney (S–U): Gl, glomerulus; Rt, renal tubule; Lt, lymphoid tissue; Liver (V–X). Bars (A–C), (J–L) and (M–X) = 50 ␮m; (D–F) = 100 ␮m and
(G–I) = 500 ␮m.
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3.4. Relative expression levels of omTERT mRNA, omTERT
protein and PCNA in different tissues of whole fish
The magnitude of omTERT mRNA expression in different fish tissues was assessed by stereological analysis
(expressed in volume density (Vv ) units) and was greatest in testes (which were full of dividing sperm cyst). In
comparison, lower expression was observed in ovaries (containing few PV oocytes) while almost equivalent expression
levels were detected among the regenerative organs (i.e.,
kidney, intestines, gill and liver). On the whole, expression
of omTERT mRNA and protein, and PCNA was consistently higher in testes than in ovaries. The weakest omTERT
mRNA level was observed in muscles (Fig. 4A). In general, the Vv value for omTERT protein is greater than the Vv
value for omTERT mRNA in all tissues examined (Fig. 4B).
Interestingly, the Vv value for PCNA was very similar to
that of the omTERT protein in most fish tissues, except
in liver and muscle where the PCNA Vv value was less
(Fig. 4C).
3.5. Correlations
Pearson’s correlation analyses showed a significant positive relationsip between omTERT mRNA and omTERT protein
expression in male O. melastigma (r2 = 0.9104, p < 0.0001),
however, the relationship was poor in female O. melastigma.
There was also a statistically significant correlation between
PCNA, with omTERT mRNA as well as omTERT protein for
both male and female fish (Table 3).
3.6. Hypoxia
O. melastigma were exposed to normoxia and hypoxia for
3 months. Fish mortality in the hypoxic group was 8% and
mortality occurred mostly in the first 2 days of hypoxic exposure and no mortality was observable after 7 days. Adult male
fish were processed for ISH and IHC. As shown in Fig. 5A, a
significant induction of omTERT mRNA (p < 0.001) and a corresponding increase in omTERT protein (Fig. 5B; p < 0.01) was
observed in liver hepatocytes of hypoxic male fish as compared
Fig. 4. Volume density (Vv ) indices of (A) omTERT mRNA, (B) omTERT protein and (C) PCNA signals in different organs of male and female whole fish sections.
Values represent the mean ± SEM; n = 5.
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R.Y.C. Kong et al. / Aquatic Toxicology 86 (2008) 131–141
Table 3
Pearson correlation between (A) omTERT protein and omTERT mRNA, (B)
PCNA and omTERT protein and (C) PCNA and omTERT mRNA expression (quantified by stereological volume density) in male and female adult O.
melastigma (n = 5 fish × 6 organs)
Coefficient of determination (r2 )
(A) omTERT mRNA vs.
omTERT protein
(B) PCNA vs. omTERT protein
(C) PCNA vs. omTERT mRNA
Male
Female
0.9104***
0.0159
0.8926***
0.9233***
0.1643*
0.3609***
Significant correlation is indicated by asterisks (* p < 0.05; *** p < 0.0001).
to the normoxic control. Additionally, an increase in PCNApositive staining but reduced TUNEL (apoptotic) activity was
also observed in the liver of hypoxic fish (Fig. 5C and D;
p < 0.01).
4. Discussion
The fixation and processing protocol developed for adult
whole O. melastigma in this study is reproducible and costeffective for ISH and IHC analyses of the omTERT gene and
protein, PCNA and TUNEL assay in multiple organs of a single fish. Applying these techniques to O. melastigma, we have
demonstrated the feasibility of simultaneous expression and
quantification analyses of multiple genes/proteins in whole fish
sections without needing to dissect out individual organs for tissue processing, thereby facilitating high throughput molecular
analyses in a small fish.
Applying the ISH and IHC analyses on serial sections of O.
melastigma, we further demonstrated that it is possible to simul-
139
taneously localize and correlate mRNA and protein expression in
different fish tissues, as well as analyse the nucleocytoplasmic
distribution of target protein molecules in specific cell types;
which is not easily achievable by other molecular techniques.
For instance, in previtellogenic oocytes, omTERT mRNA and
protein were separately localised in the cytoplasm and nucleus,
respectively (Fig. 3D and E), while in renal epithelial tubules and
enterocytes of the intestinal mucosa, omTERT mRNA and protein were found in both the nuclear and cytosolic compartments.
Alternatively, PCNA immuno-reactivity was confined largely to
the nucleus of proliferative cells such as dividing sperm cells
in testes, granulosa cells in ovarian follicles, neuronal cells in
proliferative zones of the brain, epithelial cells of gill filaments,
proliferative cells in the crypt region of mucosal folds and lymphoid tissues in kidneys. Although omTERT immunoreactivity
was detected by IHC in fish ovary, kidney and intestines, Western
blot analysis failed to detect any omTERT protein band of the
expected size (ca.120 kDa) in either the nuclear or cytoplasmic
fractions of these tissues. This could be due to low endogenous levels of the omTERT protein in these tissues that may be
associated with factors such as batch variation (e.g. fewer PV
oocytes in ovaries of female at late stages of oogenesis) or quality of protein extracts (e.g. contaminants in intestinal lumen).
Nonetheless, the above findings indicate that IHC is a highly
sensitive, reproducible and effective method for detection of
omTERT protein in multiple tissues in vivo. Conceivably, the
techniques developed in this study could be easily adapted to
study in vivo expression of other genes and/or changes in cellular proteins in response to various stressors/toxicants in the
marine environment.
Stereological analysis of ISH signals as represented by
volume density (Vv ) indices demonstrated similar patterns of
Fig. 5. Volume density (Vv ) indices of (A) omTERT mRNA, (B) omTERT protein, (C) PCNA and (D) TUNEL signals in liver hepatocytes of male O. melastigma
after chronic exposure to hypoxia (1.8 ± 0.2 mg O2 L−1 ) for 12 weeks. Values represent the mean ± SEM; n = 3 per treatment group. Values of hypoxia exposure
groups that are significantly different from the normoxia controls (6.4 ± 0.2 mg O2 L−1 ) are indicated by asterisks (** p < 0.01, *** p < 0.001).
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omTERT mRNA distribution among organs of O. melastigma,
relative to that measured by real-time RT-PCR in a previous
study (Yu et al., 2006), whereby the level of gene expression was found to occur in the order: gonads regenerative
tissues > livers, muscles. Likewise, quantification of the IHC
results also revealed differential expression levels of the
omTERT protein and PCNA in different organs of adult O.
melastigma. Correlations between the Vv indices of omTERT
mRNA and omTERT protein in O. melastigma tissues were statistically significant in male fish (r2 = 0.9104, p < 0.0001), but
no correlation was observed in female fish, suggesting a sexspecific relationship between omTERT mRNA and protein in
O. melastigma. This, however, should be further confirmed by
studying more female individuals to allow correlation analysis
at the organ level. The PCNA Vv indices were positively related
to the omTERT mRNA and protein Vv indices in tissues of both
male and female O. melastigma (Table 3), indicating that the possible involvement of omTERT expression in cell proliferation is
not limited only to mammalian cancer cells in vitro (Greider,
1998; Smith et al., 2003), but is also pertinent for non-tumor
fish cells in vivo. Our present findings show that whole adult O.
melastigma provides a sensitive and versatile in vivo system for
studying omTERT expression in multiple tissues/cells at different developmental and reproductive stages, which would help
to provide further insights about the cellular role of TERT in
growth, aging, reproduction and carcinogenesis, and its utility
in environmental toxicology.
A previous study using real-time RT-PCR (Yu et al., 2006)
showed that exposure of O. melastigma to hypoxia for 96 h
resulted in upregulated expression of omTERT mRNA in the
fish liver. In the present study, using quantitative ISH (Vv
index), we have also shown significant induction of omTERT
mRNA in liver of male O. melastigma after a 12-week exposure
to chronic hypoxia. This suggests that, unlike other hypoxiainducible genes (e.g. EPO and VEGF), in vivo expression of
the omTERT gene in hypoxic livers of O. melastigma is a persistent, rather than a transient response. Induction of omTERT
protein in the fish liver was accompanied by a concomitant
and significant increase in PCNA and a reduction in TUNEL
apoptotic activity. The PCNA and TUNEL results suggest a
possible involvement of omTERT in the regulation of hepatocyte turnover in O. melastigma. It must be emphasized also that
the same set of O. melastigma serial sections that were used for
omTERT mRNA, omTERT protein, PCNA and TUNEL analyses
in liver can be further quantified for similar molecular changes
in other organs, and would therefore allow direct comparison
of organ-specific molecular changes in response to hypoxic
stress.
In conclusion, the results of this study suggest that O.
melastigma can potentially serve as a good marine fish model
for ecotoxicological studies. The fixation and processing protocol we have developed for this whole fish system allows high
throughput and concomitant in vivo quantification of a variety
of target mRNA and proteins in different subcellular compartments and tissues in a single fish. This system confers significant
advantages in studying in vivo molecular responses to a variety
of toxicants/stresses in the marine environment.
Acknowledgements
This study was supported by a grant from the University
Grants Committee of the Hong Kong Special Administrative
Region, China (Project no. AoE/P-04/04) and CityU grants
(Project nos. 7001834 and 7002117). The research was also
supported, in part, by a grant from the US EPA STAR grant
no. R-831846. The authors would like to thank Prof. Juro
Koyama, Kagoshime University, Japan, for introducing us the
O. melastigma.
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