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European Journal of Histochemistry 2010; volume 54:e16
[page 74] [European Journal of Histochemistry 2010; 54:e16]
Immunohistochemical
localization of IGF-I, IGF-II
and MSTN proteins during
development of triploid sea
bass (
Dicentrarchus labrax
)
G. Radaelli,1C. Poltronieri,1
C. Simontacchi,1E. Negrato,1
F. Pascoli,1A. Libertini,2D. Bertotto1
1Department of Experimental Veterinary
Sciences, University of Padua, Italy
2Institute of Marine Science (ISMAR),
Venice, Italy
Abstract
The cellular localization of IGF-I, IGF-II and
MSTN proteins was investigated during onto-
genesis of triploid sea bass (Dicentrarchus
labrax) by an immunohistochemical approach.
The results were compared with those
observed in diploids. IGF-I immunostaining
was mainly observed in skin, skeletal muscle,
intestine and gills of both diploids and
triploids. From day 30 of larval life, IGF-I
immunoreactivity observed in skeletal muscle,
intestine, gills and kidney was stronger in
triploids than in diploids. At day 30, triploids
exhibited a standard length significantly high-
er than the one of diploids. Although IGF-II and
MSTN immunoreactivity was detectable in dif-
ferent tissues and organs, no differences
between diploids and triploids were observed.
The spatial localization of IGF-I, IGF-II and
MSTN proteins detected in this study is in
agreement with previous findings on the dis-
tribution of these proteins in diploid larvae and
fry. The highest IGF-I immunoreactivity
observed in triploids suggests a possible
involvement of ploidy in their growth perform-
ance.
Introduction
In the last years, the use of chromosome set
manipulation techniques to induce triploidy
has been employed to increase growth per-
formance in both freshwater and marine fish
species.1-5 Although triploids are morphologi-
cally similar to diploids, they are functionally
sterile and may give economical advantage of a
potential faster post-puberal growth.6
However, comparing the growth performance
between triploid and diploid animals, different
results have been observed in relation to the
species examined, the age and the rearing
conditions.7The European sea bass (D. labrax)
is a marine teleost fish of great interest in
aquaculture, due to its meat quality and the
large size that it can reach. Sea bass repre-
sents one of the numerous marine species that
has been subjected to triploidization3,8-9 in
order to study the growth performance.7By
evaluation of body weight, Felip et al.3,10
observed that in juveniles, diploids reached
similar growth rates as those of triploids,
whereas adult triploids grew slower than adult
diploids although they exhibited similar fork
length. No papers report on the cellular local-
ization of growth factors such as IGFs and
MSTN during ontogenesis of triploid fish.
It is well known that a complex of endo-,
para- and autocrine ways of action regulates
growth. In fish, the Insulin-like Growth Factor
(IGF) complex and myostatin (MSTN) play a
key role in the growth regulation.11-14 The IGF
complex includes the two highly conserved
primary ligands, IGF-I and IGF-II, high-affini-
ty transmembrane receptors that belong to
the insulin/IGF receptor family and six IGF-
binding proteins (IGFBP-1 to -6).15-17 IGF-I is
produced mainly in the bony fish liver,
although numerous other organs express this
molecule as well.16,18-28 IGF-II shows structural
sequence similarity to IGF-I and in fish
exhibits an ubiquitous expression, where it
acts mainly as a growth factor.21,25,27,29-40
Myostatin (MSTN) is a member of the TGF-β
superfamily and in fish its expression has
been observed in several organs such as
brain, eyes, exocrine and endocrine pancreas,
gills, gonads, heart, intestine, kidney, liver,
oesophagus, pharynx, skin, spleen, stom-
ach12,41-53 and muscle fish explants.54
Since the data concerning the growth per-
formance of triploids are mainly focused on
body weight and fork length, the aim of this
study was to evaluate whether diploid and
triploid sea bass (D. labrax) differed in terms
of immunohistochemical localization of IGF-I,
IGF-II and MSTN proteins. For this purpose,
the cellular sites of protein distribution were
examined during development and growth in
larvae and fry of both diploid and triploid ani-
mals.
Materials and Methods
Animals
Sea bass (D. labrax) eggs and milt were
obtained from broodstock held in a fish farm at
Pellestrina (Venice, Italy). Diploids and
triploids were obtained as described by
Colombo et al.8Briefly, females were stimulat-
ed with a single dose of 10 µg kg–1 of GnRH
analogue des-Gly10, [D-Ala6]-LH-RH ethylamide
(Sigma, USA). Males were not hormonally
treated. Nearly 50-60 h after hormone treat-
ment, fish were netted, anaesthetized and
gametes were collected by a gentle abdominal
compression in both males and females.
Within 5 min from fertilization, eggs were rap-
idly rinsed from sperm and submitted to a cold
shock (0-2°C) for 20 min to prevent the extru-
sion of the second polar body. An equivalent
part of fertilized egg mass was untreated and
used as diploid control. Ploidy of animals was
determined by flow-cytometry assessment of
the nuclear DNA content in in toto larvae.55
Diploid and triploid eggs were reared, till
hatch, into two different 80 L incubators, under
the same hydrological conditions (temperature
range:15.5-22.8°C). Larvae hatched in 36 h
and, after yolk sack absorption, were trans-
ferred into two separate 1 m3tank and fed
accordingly to rearing protocol. At 2, 6, 10, 30,
45, 60, 74 days post hatch (dph) a pool of lar-
vae from each ploidy was sampled and
euthanasized with a lethal dose of MS-222
(Sandoz, Italy).
Growth performance
At each age class (except at 2 dph), the stan-
dard length (LS, the distance from snout to the
tip of the notochord or hypural plate) of 10-12
larvae or juveniles per ploidy was measured
under a binocular microscope.
Fixation and embedding
Animals were fixed in 4% paraformaldehyde
prepared in phosphate-buffered saline (PBS,
Correspondence: Giuseppe Radaelli, Department
of Experimental Veterinary Sciences, University
of Padua, Italy.
Tel: +39.049.790165, Fax: +39.049.641174.
E-mail: giuseppe.radaelli@unipd.it
Key words: IGF-I, IGF-II, MSTN, immunohisto-
chemistry, triploid.
Acknowledgements: the authors wish to thank
the team of the Pellestrina fish farm (Veneto
Agricoltura, Italy). This research was supported
by grants from the Italian Ministero
dell’Università e della Ricerca Scientifica e
Tecnologica (MIUR) and of the University of
Padua (Progetto di Ateneo).
Received for publication: 23 December 2009.
Accepted for publication: 12 February 2010.
This work is licensed under a Creative Commons
Attribution 3.0 License (by-nc 3.0).
©Copyright G. Radaelli et al., 2010
Licensee PAGEPress, Italy
European Journal of Histochemistry 2010; 54:e16
doi:10.4081/ejh.2010.e16
Original paper
[European Journal of Histochemistry 2010; 54:e16] [page 75]
0.1 M, pH 7.4) at 4°C overnight, 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 micro-
tome.
Immunohistochemical procedure
All the antibodies used for this study are
detailed in Table 1. Immunohistochemical
staining was done using the Elite ABC KIT sys-
tem (Vector Laboratories, Inc., Ca, USA).
Before applying the primary antibody, endoge-
nous peroxidase activity was blocked by incu-
bating the sections in 3% H2O2in PBS. The
non-specific binding sites were blocked by
incubating the sections in normal goat serum
(Dako, Italy). Then, sections were incubated
with specific primary antisera (see Table 1)
overnight at 4°C. After washing with PBS, sec-
tions were incubated with biotin-conjugated
anti-mouse Ig antibodies (Dako), washed with
PBS and reacted with peroxidase-labeled
avidin-biotin complex (Vector Laboratories,
Inc., Ca, USA). The immunoreactive sites were
visualized using diaminobenzidine (DAB)
(Sigma, Italy) as the chromogen. To ascertain
structural details, sections were counter-
stained with Mayer’s haematoxylin.
Controls
The specificity of the immunostaining was
verified by incubating sections with: i) PBS
instead of the specific primary antibodies (see
Table 1); ii) preimmune sera instead of the
primary antisera; iii) PBS instead of the sec-
ondary antibodies and iv) by absorption of the
antisera with excess of synthetic peptides (3
μg/L) before incubation with sections. The
results of these controls were negative (i.e.
staining was abolished).
Statistical analysis
A Student’s t-test for independent samples
was used to determine any significant differ-
ences between mean LS of diploids and
triploids at each age class. Statistical signifi-
cance was taken as P<0.05.
Results
Ploidy in the examined animals
All the putative triploid sea bass were char-
acterized by 1.5 fold the nuclear DNA amount
of the diploid fish (data not shown), thus con-
firming their triploid status and the success of
triplodization.
Table 1. Antibodies used in the current study.
Antibody name and origin Immunogen Source and references Significance
and dilution for use with fish tissues
Anti-IGF-I; mouse polyclonal IGF-I from fish Perrot et al.21 Proliferation and
anti-insulin-like growth factor-I (S. aurata)1:100 differentiation of
satellite cells,
induced during
muscle regeneration
Anti-IGF-II; mouse polyclonal IGF-II from fish EuroGentec, Belgium; Proliferation of
anti-insulin-like growth factor-II (S. aurata) 1:500 Radaelli et al.35 satellite cells, induced
during muscle
regeneration
Anti-MSTN; mouse polyclonal MSTN from fish EuroGentec, Belgium; Expression of MSTN
anti-myostatin (S. aurata) 1:800 Radaelli et al.47 precursor, occurring
soon after muscle
differentiation
Table 2. Immunohistochemical localization of IGF-I in diploids (D) and triploids (T) of
sea bass.
Tissue 2 dph 6 dph 10 dph 30-45 dph 60 dph 74 dph
DT D TD TD TDTDT
Gill epithelium ** - - - -+ ++ + ++ + ++
Heart ** +/- +/- +/- +/- +/- +/- +/- +/- +/- +/-
Gut epithelium ++ +/- ++ ++ +++ ++ + ++ + ++
Liver ** +++++ +++++
Kidney ** ++++/- +/- + +/- + +/- +
Pancreas ** - - - -+/- +/- +/- +/- +/- +/-
Skin ++ ++ ++ ++++ ++ ++++
Skeletal muscle ++ + ++ ++ +++++ +/- ++++
Yolk sac ++ +/-
Staining: -, not detectable; +/-, slight but above background levels; + moderate; ++, marked staining. *Tissue not found on the sec-
tions examined at this stage.
Table 3. Immunohistochemical localization of IGF-II in diploids (D) and triploids (T) of
sea bass.
Tissue 2 dph 6 dph 10 dph 30-45 dph 60 dph 74 dph
DT D T DTD TDTD T
Gill epithelium ** - - - -+ +/- +++ +/-
Heart ** - - - -+ +/- +/- +/- +/- +/-
Gut epithelium + +/- + +/- ++ ++ +++/- +/- +/- +/-
Liver ** ++/- +/- +/- + +/- + +/- + +/-
Kidney ** +/- - +/- -- ---- -
Pancreas ** - - - - - - --- -
Skin ++ + + +++ ++++ +
Skeletal muscle ++ + + ++/- +/- +/- +/- +/- +/- +/-
Yolk sac +/- +/-
Staining: -, not detectable; +/-, slight but above background levels; + moderate; ++, marked staining. *Tissue not found on the sec-
tions examined at this stage.
[page 76] [European Journal of Histochemistry 2010; 54:e16]
Original paper
Growth performance
Mean values of LS in diploid and triploid fish
for each age are shown in Figure 1.
The statistical analysis highlighted a differ-
ence only in 30 dph fish with triploids signifi-
cantly greater than diploids (P<0.01).
Immunohistochemical localization
of IGF-I, IGF-II and MSTN proteins
General
Immunohistochemical localization of IGF-I,
IGF-II and MSTN in different tissues of
diploid and triploid sea bass is summarized in
Tables 2-4.
IGF-I
In larvae aged 2 days, IGF-I immunoreactiv-
ity was detected in the epithelia of developing
intestine and skin, as well as in lateral muscle
and yolk sac of both diploids and triploids
(Table 2). In larvae aged 6 days, skeletal mus-
cle of both diploids and triploids showed a
marked immunostaining (Figure 2A, B). At
this developmental stage, a marked immuno -
reactivity was also observed in the epithelia of
gut and skin (Table 2). In larvae aged 6-10
days, a moderate immunostaining was also
detected in liver and developing kidney, where-
as heart musculature exhibited a faint reactiv-
ity (Table 2). From day 30, skeletal muscle as
well as the epithelia of gills, gut and kidney of
triploids exhibited an immunoreactivity higher
than the one of diploids (Table 2, Figure 2C-F).
IGF-II
In both diploids and triploids aged 2 days, a
moderate IGF-II immunostaining was observed
in the epithelia of skin and developing intes-
tine (Table 3), as well as in skeletal muscle
(Figure 3A, B). At this developmental stage, a
faint IGF-II immunoreactivity was also
observed in the yolk sac (Table 3). From day 6,
IGF-II reactivity was also detected in liver of
both diploids and triploids and in developing
kidney of diploids (Table 3). From day 30, heart
musculature and gill epithelium exhibited an
IGF-II immunostaining, whereas no reactivity
was detected in kidney (Table 3). At all stages,
tissues exhibited an IGF-II immunostaining
ranging from faint to moderate, although the
gut epithelium of larvae aged 10 days showed a
marked reactivity (Table 3, Figure 3C, D). No
significant differences between diploids and
triploids were observed in immunoreactivity.
MSTN
In both diploids and triploids aged 2-10 days,
a MSTN immunostaining was observed in
skeletal muscle and in the epithelia of skin and
developing intestine (Table 4, Figure 4A-F).
From day 6, MSTN immunoreactivity was also
detected in liver, heart musculature and in
developing kidney, although the staining was
different between diploids and triploids (Table
4, Figure 4C-F). Pancreas exhibited a MSTN
immunostaining only in larvae aged 10 days
(Figure 4F). From day 30 the epithelium cover-
ing gill filament was stained, too (Table 4). No
significant differences between diploids and
triploids were observed in immunoreactivity.
Discussion
The present study reports novel information
on the cellular localization of IGF-I, IGF-II and
MSTN proteins during development and
growth of diploid and triploid sea bass (D.
labrax). Although triploids are morphologically
similar to diploids, they have larger but fewer
cells in most tissues and organs and they are
sterile, thus increasing their appealing for
aquaculture as a mean to protect somatic
growth, survival and flesh quality from the neg-
ative effects of sexual maturation.5,6,56
A general opinion is that triploids should grow
faster than diploids, since their genes are more
numerous and their cells are larger.56 However,
the literature concerning the growth performance
of triploids and their diploid counterparts is often
contradictory. Several studies have reported that
growth of triploid fish was similar to the diploids
one, during the juvenile period,3,10,57-59 whereas it
was higher at their sexual maturation time.60-63
Moreover, Razak et al.64 found that transgenic
diploids of tiliapia were superior in growth per-
formance, followed by transgenic triploids, non
transgenic diploids and non transgenic triploids.
In terms of growth performance, the literature of
triploids is mainly limited to the evaluation of
growth rate and fork length, whereas no papers
report on the cellular localization of growth fac-
tors, such as IGFs and MSTN, during ontogenesis.
In the present study the cellular localization of
IGF-I, IGF-II and MSTN proteins was studied from
hatching to juvenile stages by immunohisto-
chemistry using polyclonal antisera raised
against sea bream IGF-I,21 IGF-II35 and MSTN.47 In
general, the cellular sites of immunoreactivity
observed in triploids were identical to those found
in diploids for all tested antibodies.
Cellular localization of IGF-I
The pattern of immunostaining for IGF-I
observed in both diploids and triploids was
Table 4. Immunohistochemical localization of MSTN in diploids (D) and triploids (T) of
sea bass.
Tissue 2 dph 6 dph 10 dph 30-45 dph 60 dph 74 dph
DT DT DT D TDTD T
Gill epithelium ** - - -- + +/- + +/- + +/-
Heart ** +/- - +/- - +/- +/- +/- +/- +/- +/-
Gut epithelium ++ +/- + ++ +/- ++++++
Liver ** ++/- +/- - +/- - +/- - +/- -
Kidney ** +/- - +/- - +/- - +/- +/- +/- +/-
Pancreas ** - - +/- +- ---- -
Skin ++ ++ ++ + +/- + +/- + +/-
Skeletal muscle ++ ++ +/- +/- + +/- +/- +/- + +/-
Yolk sac + +/-
Staining: -, not detectable; +/-, slight but above background levels; + moderate; ++, marked staining. *Tissue not found on the sec-
tions examined at this stage.
Figure 1. Mean stan-
dard length of
diploid and triploid
fish at each age class.
Vertical bars repre-
sent standard errors
of the mean and
asterisks indicate sig-
nificant difference
between groups.
similar to that observed in diploids of S. aura-
ta,21 U. cirrosa,23,27 D. labrax,28 demonstrating
that the sequence similarity between IGF-I
from different fish species is sufficient to
allow cross-species immunoreactivity. A simi-
lar pattern of immunostaining was also
observed in O. niloticus by Berishvili et al.26
During early larval life, IGF-I immunoreactivi-
ty was mainly detected in the epithelia of gut,
skin and kidney, as well as in skeletal muscula-
ture and liver. No significant differences
between diploids and triploids were observed in
immunoreactivity. Interestingly, from day 30,
skeletal muscle and the epithelia of gills, gut and
renal tubules of triploids exhibited an immunos-
taining higher than that of diploids. In our
experimental conditions, the growth of triploids
measured in terms of standard length was simi-
lar to that of diploids, although at day 30 triploids
exhibited a significantly higher length. As men-
tioned above, triploid fish have larger but fewer
cells in their tissues and the reduction in the cell
number could be compensated by a higher pro-
tein synthesis.56 In fish, body growth is mainly
correlated to that of skeletal lateral muscle,
which continues to grow significantly even into
juvenile life in many species, through continu-
ous hyperplasia and hypertrophy.65 Johnston et
al.66 found that triploid Salmonidae exhibited a
reduced hyperplasia as a consequence of a
decrease in the satellite cell number. They sug-
gested that the reduced number of myocytes
detected in triploids was compensated by their
rates of hypertrophic growth, greater than the
diploids ones.66
Moreover, Alonso et al.67 observed that dur-
ing fin regeneration of O. mykiss, the protein
synthesis (per unit muscle mass) was higher
in triploids than in diploids.
Cellular localization of IGF-II
In our previous immunohistochemical
study, the use of an antiserum raised against
sea bream IGF-II gave us the possibility to
detect the cellular distribution of IGF-II protein
in D. labrax28, as well as in U. cirrosa,27 demon-
strating that there is a high degree of similar-
ity between IGF-II proteins from different fish
species and therefore allows cross-species
immunoreactivity.
In young larvae, IGF-II immunostaining was
found in skeletal muscle, liver, the epithelia of
skin and developing intestine of both diploids
and triploids, as well as in the epithelium of
renal tubules of diploids. From day 30, heart
musculature and the epithelium of gill fila-
ments exhibited an IGF-II immunostaining.
The pattern of IGF-II immunopositivity was
similar to the one observed in diploids of sev-
eral fish species.21,27,35,40 However, in the present
work we did not observe significant differ-
ences between diploids and triploids in terms
Original paper
[European Journal of Histochemistry 2010; 54:e16] [page 77]
Figure 2. Immuno-histochemical localization of IGF-I in sea bass larvae and fry. All pan-
els are counter-stained with haematoxylin. A, C, E: diploid animals; B, D, F: triploid ani-
mals. A Sagittal section of a 6-day larva. A marked IGF-I immunostaining is present in the
trunk musculature. B Transverse section of a 6-day larva. A marked IGF-I immunostain-
ing is present in the trunk musculature, intestine (I) and skin (arrow). C-D Sagittal sec-
tions of 45-day larvae. Skin epithelium (asterisks) exhibits a marked positivity in both
diploids and triploids. An immunostaining is also present in skeletal muscle (M),
although in triploids (D) the reactivity is stronger than in diploids (C). Cartilage (c) is
negative. E-F Gills of 60-day fry. Immunostaining is present in the epithelium of the gill
filaments and the reactivity is stronger in triploids (F) than in diploids (E). Bars (A) 20
μm, (B) 20 μm, (C) 15 μm, (D) 15μm, (E) 12.5 μm, (F) 12.5 μm.
Figure 3. Immunohistochemical localization of IGF-II in sea bass larvae. All panels are
counterstained with haematoxylin. A, C: diploid animals; B, D: triploid animals. A-B
Sagittal sections of 2-day larvae. In both diploids and triploids a moderate immunostain-
ing is present in skeletal muscle and skin (arrows). C-D Sections of 10-day larvae. In both
diploids and triploids a marked immunostaining is detectable in the intestinal epithelium
(I). Bars (A) 15 μm; (B) 20 μm, (C) 20 μm, (D) 12.5 μm.
[page 78] [European Journal of Histochemistry 2010; 54:e16]
of IGF-II immunoreactivity, suggesting that a
similar amount of IGF-II protein is detectable
in the tissues of both diploids and triploids.
Cellular localization of MSTN
The high degree of similarity between
MSTNs from different fish species allowed us
to use an antiserum raised against sea bream
MSTN to detect the cellular distribution of the
protein in different fish species, including D.
labrax.28,40 In the present work, the pattern of
MSTN immunostaining was similar to the one
observed for IGF-I and IGF-II. A co-localization
of IGF-I, IGF-II and MSTN proteins has been
observed in cultured muscle explants from S.
aurata,54 suggesting an autocrine-paracrine
action of these factors in regulating develop-
ment and growth of fish. As for IGF-II, we did
not observe significant differences between
diploids and triploids in terms of MSTN
immunoreactivity, suggesting that a similar
protein amount is present in the tissues of
both diploids and triploids.
The results we report here show for the first
time the immunohistochemical localization of
IGF-I, IGF-II and MSTN proteins during ontoge-
nesis of diploids and triploids of D. labrax. The
spatial localization of all examined growth fac-
tors is identical in both diploids and triploids
although from day 30, skeletal muscle, as well
as the epithelia of gills, gut and kidney of
triploids exhibited an IGF-I immunoreactivity
higher than the one of diploids. Interestingly,
at day 30, the standard length of triploids was
significantly higher than the diploids one. We
intend to further investigate, by Real-Time
PCR, the IGF-I espression in these tissues, in
mind to better understand whether triploidy
can result in any growth advantage.
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