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First published online December 14, 2007
Journal of Experimental Biology 211, 128-137 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.006890
Myogenesis and muscle metabolism in juvenile Atlantic salmon (Salmo salar) made transgenic for growth hormone
1 Department of Biology and Centre for Advanced Research in Environmental
Genomics, University of Ottawa, PO Box 450, Stn A, Ottawa, Ontario, Canada,
K1N 6N5
2 Ocean Sciences Centre, Memorial University of Newfoundland, and AquaBounty
Technologies Inc., St John's, Newfoundland, Canada, A1C 5S7
Author for correspondence (e-mail:
tmoon{at}uottawa.ca)
Accepted 20 October 2007
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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Key words: myosatellite cells, metabolism, enzymes, myogenin, MyoD I, MyoD II, RT-PCR
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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) and are capable of reaching market size 1 year earlier than
standard salmon currently cultured in Atlantic Canada
(Fletcher et al., 2004;
Deitch et al., 2006). The
mechanism(s) responsible for such rapid growth in transgenic salmon especially
at the very early stages (0–4 months) has yet to be resolved
(Hill et al., 2000). A recent
study using transgenic coho salmon (Oncorhynchus kisutch) points to
their enhanced appetite as one possible explanation for the accelerated growth
(Stevens and Devlin, 2005).
However, it may be that the enhanced appetite is driven by the increased
anabolic effects of GH on protein synthesis and growth of skeletal muscle and
other tissues (Fauconneau et al.,
1996). A number of studies have demonstrated that both juvenile
and adult GH transgenic salmon have higher metabolic rates than
non-transgenics (Stevens et al.,
1998; Cook et al.,
2000; Lee et al.,
2003; Deitch et al.,
2006).
Fish growth is generally indeterminate, with most species continuing to
grow in mass and length throughout their life. Since skeletal muscle comprises
a large proportion of body weight, growth in body mass is to a large extent
dependent upon increased muscle mass
(Weatherly and Gill, 1987;
Houlihan et al., 1988).
Post-larval growth of skeletal muscle is attributed to the proliferation and
differentiation of muscle satellite stem cells or myosatellite cells (MCs)
(Fauconneau and Paboeuf,
2001). These small, spindle-shaped cells have heterochromatic
nuclei and are located between the sarcolemma and the basal lamina of
differentiated muscle fibres (Koumans et
al., 1990). During muscle growth, MCs either fuse with
pre-existing muscle fibres resulting in hypertrophic growth or fuse together
to form myotubes that differentiate into new fibres in hyperplastic growth
(Allen et al., 1979;
Veggetti et al., 1990;
Koumans et al., 1993;
Johnston et al., 1995).
Proliferation and differentiation of MCs are regulated by transcription
factors called myogenic regulatory factors (MRFs)
(Hawke and Garry, 2001). MyoD
and myogenin are helix–loop–helix transcription factors that play
an essential role in muscle cell determination, proliferation and
differentiation (Feldman and Stockdale,
1991) during development and growth of vertebrates including fish
(Xie et al., 2001). These two
factors together with Myf5 and MRF4 comprise the MRF family
(Watabe, 2001). In addition to
transcription factors, hormones including but not restricted to GH, insulin
and insulin-like growth factor-I (IGF-I) are implicated in vertebrate muscle
growth (Mommsen and Moon,
2001). Circulating levels of these hormones are affected by ration
size, and the composition and energy status of the fish
(Mommsen and Moon, 2001).
Skeletal muscle consists of a mixture of small and large diameter fibres,
and it is generally believed that the smaller fibres are earlier stages of the
larger ones, and as such are diagnostic of hyperplastic growth
(Weatherly and Gill, 1987).
This is supported by the observation that a reduction occurs in the proportion
of small muscle fibres with increasing length/weight of the fish
(Stickland, 1983;
Koumans and Akster, 1995).
Several studies have demonstrated that GH administration increases the
proportion of small diameter fibres in rainbow trout (Oncorhynchus
mykiss), suggesting increased hyperplasia (Weatherly and Gill, 1982;
Fauconneau et al., 1997). A
study of the white muscle composition revealed that GH transgenic coho salmon
fry had a greater proportion of small muscle fibres relative to
non-transgenics, thus supporting the hypothesis that GH promotes muscle
hyperplasia (Hill et al.,
2000). A more recent study of GH transgenic coho salmon suggests
that the accelerated muscle growth is the result of decreased myostatin
expression, a negative regulator of muscle growth
(Roberts et al., 2004).
The goal of this study was to determine whether the ectopic expression of
GH in transgenic Atlantic salmon influences the in vivo and in
vitro proliferation and differentiation of skeletal muscle MCs.
Proliferation was assessed by bromodeoxyuridine (BrdU) incorporation and
differentiation was assessed by quantifying the expression of the MRFs MyoD I,
MyoD II and myogenin. In view of the elevated metabolic rate reported for
GH-transgenic salmon (Stevens et al.,
1998; Cook et al.,
2000; Deitch et al.,
2006), the activities of a number of metabolic enzymes were also
investigated to determine whether enzyme activities correlated with growth
rate.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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Experimental animals
The transgenic Atlantic salmon strain (termed EO-1
) used in this
study is hemizygous for a single functional ectopic copy of a GH transgene
(Fletcher et al., 2004;
Yaskowiak et al., 2006). This
strain was created in 1989 by injecting fertilized eggs with a chimeric GH
gene construct (opAFP-GHc2) consisting of a Chinook salmon (Oncorhynchus
tshawytscha) GH cDNA regulated by an ocean pout (Macrozoarces
americanus) antifreeze protein gene promoter.
The salmon selected for this study were from the 5th generation (F5) of
this lineage. The transgenic parents had been cultured in fresh water under
ambient temperatures and photoperiod. Control, wild-type salmon were collected
from the North East River, Placentia, NF (Canada), in November 2002. The F5
transgenic offspring were obtained by fertilizing a pool of eggs from seven
control (wild-type) females with milt from one GH transgenic male that was
hemizygous for the transgene. Fifty per cent of the offspring from this cross
inherited the GH transgene in accordance with simple Mendelian inheritance
rules. The non-transgenic salmon used as comparators in this study were
obtained by crossing the same pool of eggs from non-transgenic females with
sperm from a single wild-type non-transgenic male. Eggs were incubated at
6–7°C in a re-circulating vertical stacked tray system at pH
6.8–7. Approximately 1000 fry were transferred to re-circulating rearing
tanks (
1 m3) on March 3, 2003. The water pH was maintained at
6.8–7 and the temperature held at 13.0–13.8°C. All offspring
were raised in the same tank under natural photoperiod. The sample of fish
(transgenics and non-transgenics) selected for analyses at 4 and 7 months
after first feeding were representative of the modal size range of the total
population under culture. Fin clips were removed from all fish sampled, frozen
in liquid nitrogen and stored in a –80°C freezer until analysis by
PCR for the presence or absence of the GH transgene (see
Deitch et al., 2006)
(Fig. 1).
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). The mean fish
mass at the beginning of the experiment was estimated at 0.5 g for both
controls (non-transgenics) and GH-transgenics. All experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care for the use of animals in teaching and research.
Myosatellite cell isolation and culture
MCs were isolated from salmon white muscle using the standard procedures
developed by Fauconneau and Paboeuf
(Fauconneau and Paboeuf, 2000)
with minor modifications. Briefly, Atlantic salmon were killed by a blow to
the head, rinsed with cold water to remove excess mucus and disinfected by
washing with a bleach solution (1/2000) followed by phosphate-buffered saline
(PBS) and 70% ethanol. White muscle was filleted from both sides of the fish
inside a laminar flow hood at room temperature and collected in basal medium
(DMEM, Dulbecco's modified Eagle's medium with 9 mmol l–1
NaHCO3, 20 mmol l–1 Hepes and an antibiotic
cocktail – A5955, Sigma-Aldrich, St Louis, MO, USA). Muscle pieces were
then incubated for 1 h at 4°C in basal medium with 15% horse serum and a
4-fold antibiotic concentration. Muscle pieces were then chopped into small
cubes and transferred into fresh basal medium containing 1-fold antibiotic
concentration and cell extraction proceeded at room temperature. MCs were
cultured at 18°C in DMEM supplemented with 10% fetal calf serum and the
cocktail of antibiotics at 3x105 cells cm–2,
in either sterile 24 well plates or 75 cm2 flasks.
MC proliferation was assessed by BrdU incorporation
(Valente et al., 2002). BrdU
was added to the cell culture medium at a concentration of 10 µmol
l–1. Incorporation of BrdU into the MCs was measured after 24
h using an immunofluorescence kit (Roche Diagnostic, QC, Canada). Total nuclei
were stained using Hoechst 33258 dye solution (355 nm/450 nm). Total nuclei
and nuclei that incorporated BrdU were counted using the Image J 1.33 program
(National Institutes of Health, Rockville, MD, USA) from computer-generated
pictures taken with a fluorescent microscope (Axiophot, Carl Zeiss,
Oberkochen, Germany). Three random fields on each slide were counted and each
field contained at least 100 cells. Duplicate slides were made for each time
point and experiments were repeated 3 times giving a sample size of 3 using
different pools of white muscle.
In addition to comparing MC proliferation rates in white muscle isolated from GH-transgenic and non-transgenic salmon, recombinant trout GH actions were assessed at concentrations of 10 and 50 ng ml–1 (GroPep, Adelaide, Australia, GHBU020 lot: IJH-GHB1) on MCs isolated from 7 month old non-transgenic salmon white muscle. Cells were cultured for 24 h in serum replacement medium (Sigma, S0638) in 24 well plates at an initial density of 3x105 cells cm–2. Proliferation was assessed using BrdU as described above.
Myogenesis
Myogenesis was assessed by using RT-PCR to quantify mRNA levels of the MRFs
MyoD I, MyoD II and myogenin in freshly isolated muscle tissue. Since salmon
have two homologous, non-allelic MyoD genes, MyoD I and MyoD II
(Watabe, 2001), the expression
levels of both were assessed.
Total RNA was isolated from red and white muscle tissues using TRIzol® reagent (Invitrogen, Burlington, ON, Canada) in accordance with the manufacturer's instructions. Red muscle could not be isolated from the 4 month non-transgenic salmon as red muscle and white muscle are indistinguishable at this age. Muscle samples were removed immediately after the salmon had been killed, frozen on dry ice and stored at –80°C. Muscle tissue was powdered with a mortar and pestle in liquid nitrogen and RNA extracted using 1 ml TRIzol per 65–85 mg tissue. All RNA samples were run on a gel to ensure integrity. Following isolation, total RNA was treated with DNase I (Invitrogen, 18068-015), transformed into cDNA using a reverse transcriptase (Invitrogen, MMLV reverse transcriptase 28025-013) and amplified using Taq polymerase (Invitrogen, 1803842) according to the manufacturer's instructions. A control using water rather than reverse transcriptase was incorporated into the PCR reaction to ensure the absence of DNA contamination.
Specific primers were designed for MyoD I, MyoD II, myogenin and 18S using
sequences available on GenBank (accession nos AJ557148, AJ557149, AJ534875 and
AJ427629, respectively). The primer sets used are presented in
Table 1. A cycle gradient curve
was performed for each gene in each tissue to select the number of cycles
necessary to amplify the myogenic factors and the control gene according to
Mimeault et al. (Mimeault et al.,
2005; Mimeault et al.,
2006) (based on the linear phase of amplification for which the
R2 varied between 0.96 and 0.99). MyoD I, myogenin and 18S
were amplified for 25, 26 and 11 cycles, respectively, for white muscle tissue
RNA. MyoD I, MyoD II, myogenin and 18S were amplified for 26, 28, 24 and 11
cycles, respectively, for red muscle tissue RNA. The PCR conditions used were
94°C for 45 s, 59°C for 30 s and 72°C for 45 s, except for
myogenin, where the annealing temperature was 62°C rather than 59°C.
For each gene, increasing amounts of cDNA were amplified at the selected cycle
number in order to verify the linearity of the analysis; amplification was
shown to be linear for each primer (R2 varied between 0.97
and 0.99). The cycle gradient and cDNA amount studies used a pool of control
and transgenic cDNA for each muscle type run separately.
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The PCR amplification products were electrophoresed on a 1.5% agarose gel containing ethidium bromide. PCR products from each gene of interest were mixed with the PCR product of the control gene in equal amounts and run on the same gel. The gel was scanned under UV light and the density of the bands quantified using the Quantity one computer program (BioRad, Hercules, CA, USA). Data are expressed as a ratio of the band density for the gene of interest to the band density for 18S from the same tissue. In order to ensure that the PCR products corresponded to the mRNA of interest, each amplification product was cloned using a Topo cloning kit and One Shot Topo II vector (Invitrogen, K4550-01). Plasmid DNA containing the insert of interest was then purified using the QIAprep spin miniprep kit protocol (Qiagen, 27104) and sequenced by the Core DNA Facility Centre (Ottawa, ON, Canada).
Enzyme activities
Enzyme activities were determined in liver, intestine, red muscle and white
muscle tissues from transgenic and non-transgenic salmon at 4 and 7 months of
age; red muscle could not be recovered from 4 month non-transgenics. Red
muscle and white muscle were always taken from the same side and in the same
region just below the dorsal fin for all fish. Tissues were immediately frozen
on dry ice and held at –80°C until analyses were performed. Tissues
were powdered by grinding with a mortar and pestle in liquid nitrogen. The
powder was homogenized (1:5 w/v) in a glycerol-containing buffer (see
Moon and Mommsen, 1987) and
centrifuged for 10 min at 4°C and 10 000 g (Micro Centaur,
Sanyo, VWR Scientific); the resulting supernatant was used immediately for
enzyme analyses. Enzyme activities were measured under saturating substrate
conditions using standard procedures (Moon
and Mommsen, 1987). Reaction rates were assayed
spectrophotometrically at 340 nm following the appearance or disappearance of
NAD(P)H for all enzymes except citrate synthase (CS) where the oxidation of
DTNB was monitored at 412 nm. The extinction coefficients used for NAD(P)H and
DTNB were 6.22 and 13.61 (mmol l–1)–1
cm–1, respectively. Enzyme activities were measured at room
temperature (22°C) using a plate reader (Spectra Max Plus 384; Molecular
Devices, Sunnyvale, CA, USA) and SOFTmax Pro software to calculate activities.
Tissue protein content was measured using the bicinchononic acid (BCA) method
(Sigma-Aldrich) with bovine serum albumin as a standard. However, protein
content between groups changed significantly, so enzyme activities were
expressed as units (µmol min–1) of activity per g
tissue.
Statistics
Statistical analyses were performed using InStat 3 (for normality testing)
and SYSTAT (version 10). Two-way analyses of variance (ANOVA) were performed
on enzyme activities and myogenic factor expression, except for red muscle
where a one-way ANOVA was used as the 4 month non-transgenic group was absent.
When the one-way ANOVA showed significant effects, multiple mean comparisons
were made using the Tukey–Kramer comparison. Data were transformed to
obtain normality (Kolmogorov–Smirnov test) when necessary. When it was
not possible to obtain normality, non-parametric analyses
(Kruskall–Wallis) were employed. Simple correlations using SYSTAT were
performed between enzymes and fish mass.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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The specific growth rates of the transgenic salmon were approximately
double those of the non-transgenics over the first 4 months of growth
(Table 2). This resulted in the
transgenics being approximately 5 times the weight of the non-transgenics at 4
months after first feeding. Over the 4 to 7 month period, growth rates of the
transgenic fish declined significantly while those of the controls increased
slightly (Table 2). Body mass
of the 7 month non-transgenic salmon did not differ significantly from that of
the 4 month transgenics. Thus the 7 month non-transgenic salmon meet the
criteria of being appropriate weight-matched controls for the 4 month
GH-transgenics (Pitkänen et al.,
2001).
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MC yield was significantly affected by the age of the fish, with a significantly higher yield from both transgenic and non-transgenic salmon at 4 than at 7 months (P<0.001; Table 2). In addition, despite the fact that the 4 month GH-transgenics were similar in weight to the 7 month non-transgenic salmon, the yield of MCs was generally greater (Table 2).
White muscle MC proliferation in vitro
Effects of GH transgene on cell proliferation
The two-way ANOVA performed on MC proliferation rates showed an effect of
days after plating (P<0.001), an effect of presence of the
transgene (P<0.001) and an effect of the interaction
(P=0.003). The comparison using Bonferroni indicated that in
vitro MC proliferation rates differed between the 4 month transgenic and
the 7 month non-transgenic salmon (Fig.
2). No significant differences existed between proliferation rates
of the 4 month transgenic and non-transgenic or between the 4 and 7 month
non-transgenic salmon. Initial proliferation rates were approximately 10% per
day for all groups but rates increased more rapidly for the transgenic than
the non-transgenic salmon MCs before reaching a plateau of 80% per day after 6
days. Proliferation rates for the non-transgenic salmon MCs were lower, with
the 4 month group achieving a plateau by 8 days at 65% and the 7 month group
also reaching 65% by 9 days but never achieving a plateau.
Effects of growth hormone on cell proliferation
Proliferation rates of MCs isolated from 7 month non-transgenic salmon
after 24 h of culture was 10% per day (Fig.
3), a value noted in the previous experiment for this group
(Fig. 2). The addition of
recombinant trout GH to the culture media doubled cell proliferation rates
over the 24 h culture period at both GH concentrations tested
(Fig. 3).
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White muscle myogenin mRNA expression was significantly affected by the age of the fish (P<0.001) and the interaction between age and presence of the transgene (P=0.002) but not by the presence of the transgene (P=0.528). Relative myogenin mRNA was lower in 4 month transgenics compared with non-transgenic salmon of the same age (4 months) and mass (7 months; Fig. 5).
Red muscle MyoD I, MyoD II and myogenin mRNA expression were significantly higher in transgenic salmon at 7 months (P<0.001) compared with non-transgenic salmon of the same age and transgenic salmon at 4 months (Figs 6 and 7).
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Tissue enzyme activities
White muscle protein content, pyruvate kinase (PK), aspartate
aminotransferase (AST) and malate dehydrogenase (MDH) were positively
correlated with body mass (Table
3, Fig. 8). White
muscle protein content and activities of alanine aminotransferase (ALT), MDH
and citrate synthase (CS) were significantly affected by the age of the fish.
White muscle protein content, lactate dehydrogenase (LDH) and CS were
significantly affected by the presence of the transgene. For all the enzymes
tested in white muscle, except AST and isocitrate dehydrogenase (IDH), there
was an interaction between age and the presence of the transgene
(Table 3), with enzyme
activities being higher in transgenic salmon at 4 months and lower in
transgenic salmon at 7 months compared with non-transgenic salmon. Intestinal
CS was positively correlated with fish body mass (P<0.001) and
affected by age (P=0.001) and the presence of the transgene
(P=0.012); CS activity was significantly higher in transgenic salmon
compared with non-transgenic salmon (Fig.
9).
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Red muscle PK, MDH, IDH and CS were higher in 7 month non-transgenics than in the 4 or 7 month old transgenic salmon (Table 4). There was insufficient red muscle in the 4 month non-transgenics to sample. Red muscle MDH, IDH and CS were significantly negatively correlated with fish body mass.
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Liver phosphoenolpyruvate carboxykinase (PEPCK), PK, AST, glutamate dehydrogenase (GDH), LDH and CS were all significantly affected by the age of the fish, being higher in 7 month than in 4 month old salmon. Liver protein, LDH, MDH, IDH and CS were significantly affected by the presence of the transgene, with the activity of the enzyme being lower in the transgenic compared with the non-transgenic salmon. Liver protein, PK, MDH, IDH and CS were significantly affected by the interaction between the transgene and the age of the fish (Table 5).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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During this rapid growth period the GH-transgenic salmon had higher numbers
of white muscle MCs than did the weight-matched non-transgenics. In addition,
although the 4 month transgenics had the same number of MCs per gram of white
muscle as the age-matched controls, their 5-fold greater body mass indicates
that they had a greater total number of MCs. These results suggest that MC
number in addition to other factors (e.g. humoral factors, MC sensitivities)
may contribute to the more rapid growth rates observed in the transgenic
salmon. This result contrasts with the study of Valente et al.
(Valente et al., 2002), which
reported no difference in white muscle MC yield between two strains of rainbow
trout (Oncorhynchus mykiss) that differed considerably in growth
rates.
The decline in MC number with body mass in both transgenic and
non-transgenic salmon is consistent with observations made on rainbow trout
(Greenlee et al., 1995;
Fauconneau and Paboeuf, 2001)
and common carp (Cyprinus carpio)
(Koumans et al., 1991;
Alfei et al., 1994;
Koumans and Akster, 1995).
This decrease in MC number suggests that the recruitment of new muscle fibres
declines as the salmon age and as body mass increases. However, despite the
loss in this capacity with age the 7 month transgenic salmon are potentially
as capable of recruiting new muscle fibres as the smaller non-transgenic
age-matched comparators (Table
1).
If the enhanced growth rates of the GH-transgenics are at least in part
attributable to their greater MC number, then it is likely that the time at
which this acceleration in growth is initiated following first feeding is
facilitated by the presence of greater numbers of these stem cells. MCs are
thought to originate during late embryogenesis
(Stoiber and Sänger,
1996); therefore, the increase in MC number in the transgenic
salmon should be established during this stage in development.
This study clearly demonstrates that the in vitro proliferation rate profile of the MCs from GH-transgenic salmon differed from that of the non-transgenics in four ways: (1) the daily proliferation rate began to increase earlier; (2) the daily rate of increase in proliferation was accelerated; (3) the proliferation rate reached a plateau value more quickly; and (4) the plateau value for the proliferation rate was greater. Thus, not only did the white muscle from the transgenic salmon have a greater concentration of MCs than the size-matched controls, but also the MCs themselves had a greater capacity to proliferate than did the cells from non-transgenics.
The precise mechanism(s) to explain how the ectopic expression of GH
influences MC number and/or their proliferation and differentiation is
unknown. The GH-enhanced proliferation of white muscle MCs in vitro
certainly indicates that GH can have a direct effect on these MCs as reported
by Halevy et al. (Halevy et al.,
1996) and Hodik et al. (Hodik
et al., 1997) in avian muscle and Michal et al.
(Michal et al., 2002) in
canine satellite cells. Although the levels of GH in the plasma of the
transgenic salmon have not been quantified, GH transgene mRNA is expressed in
most tissues including muscle of these transgenic Atlantic salmon
(Hew et al., 1995;
Hobbs and Fletcher, 2007).
Therefore, it is possible that GH production by the muscle tissue and/or
higher levels of circulating GH is responsible for the increased proliferation
directly or via IGF-I production in muscle as reported for mice
(Kim et al., 2005) and the
C2C12 cell line (Sadowski et al.,
2001). The absence of a dose-dependent response in MC
proliferation rate when cells were exposed to GH in vitro in our
experiment suggests that the two doses tested in our study were too high.
Similarly, there was no dose–response effect in avian satellite cells at
a GH concentration between 2 and 50 ng ml–1
(Halevy at al., 1996), and
between 10 and 75 ng ml–1 in canine satellite cells
(Michal et al., 2002). The use
of lower GH doses would be necessary to observe a dose–response
effect.
GH is reported to activate myogenesis by increasing MC fusion to existing
myotubes in vitro in mice
(Sotiropoulos et al., 2006),
and MC proliferation in mice (Kim et al.,
2005), mammalian cell lines
(Sadowski et al., 2001) and
birds (Halevy et al., 1996).
It is generally believed that GH acts on white muscle through circulating or
local production of IGF-I (Kim et al.,
2005), and it was shown in rodents that IGF-I increased both MC
proliferation and MC differentiation (Allen
and Rankin, 1990; Hawke and
Garry, 2001). However, in our study the age of the fish seems to
have a greater impact than GH level, at least for MC proliferation. The fact
that differentiation is decreased in salmon white muscle at 4 months in our
experiment implicates different factors in the regulation of the expression of
myogenin mRNA.
Red muscle MyoD I, MyoD II and myogenin mRNA expression did not differ
between 4 month transgenics and their 7 month weight-matched controls,
suggesting that in red muscle the MC proliferation and differentiation rates
did not differ between transgenics and controls at this growth stage. However,
the increased level of MyoD II and myogenin expression in the 7 month old
transgenics suggests that MC proliferation and differentiation increases as
the fish increase in size. To date, nothing is known regarding the different
regulation of MyoD I and MyoD II in red muscle in adult fish; however, our
study confirms the suggestion by Delalande and Rescan
(Delalande and Rescan, 1999)
that MyoD I and II have a distinct role in red muscle.
It is apparent from the foregoing that the ectopic expression of the GH transgene in salmon altered the temporal patterns of expression of MyoD and myogenin in both white muscle and red muscle. Comparing red muscle and white muscle in transgenics, MyoD expression increased as the transgenic salmon increased in age and mass from 4 to 7 months after first feeding. This implies an increase in MC proliferation rates in both tissues over this time period. White muscle from non-transgenic salmon demonstrated the same changes in MyoD expression over the same period of time. In the case of myogenin expression, an indicator of cell differentiation, white muscle and red muscle showed increased mRNA levels as the transgenic fish aged and increased in size from 4 to 7 months. This result shows a direct link between myogenesis and fish growth rate, with MC proliferation occurring when growth rate increased.
The GH transgene also increased the activity of the glycolytic enzyme LDH
in white muscle and liver and the oxidative enzyme CS in white muscle, liver
and intestine, a result possibly reflecting a GH-mediated improvement in feed
conversion efficiency as reported for both GH-treated
(Campbell et al., 1989) and
GH-transgenic (Pursel et al.,
1990) domestic pig, and in GH-transgenic tilapia
(De La Fuente et al., 1999),
and an increase in basal metabolism of transgenic fish
(Hill et al., 2000). An effect
of the interaction between age and transgene was found in all the enzymes
assessed in white muscle, except for AST and IDH, indicating that depending on
the age of the fish the effect of the transgene was different, with higher
activity in the transgenics at 4 months and lower activity in the transgenics
at 7 months compared with the age-matched non-transgenics, except for MDH
where the opposite happens, indicating that these enzymes could be affected by
the growth rate of the fish.
The activities of all enzymes measured in the red muscle were higher in the
non-transgenic salmon at 7 months compared with both GH-transgenic groups of
salmon. This result may be explained by the higher quantity of red muscle
found in transgenic salmon compared with non-transgenic salmon
(Hill et al., 2000), resulting
in no change in the total enzyme activities per tissue. However, we did not
compare the proportion of red muscle and white muscle in our fish. Within the
liver, most of the enzymes (except ALT, glucose 6-phosphate dehydrogenase and
MDH) were affected by the age of the fish. However, PK, LDH, MDH, IDH and CS
were affected by the presence of the transgene and/or the interaction of age
and transgene. These results suggest that both oxidative and glycolytic
metabolism increase in the liver in GH-transgenic salmon at 4 and 7 months.
This statement supports previous studies reporting higher oxygen consumption
in GH-transgenic tilapia (McKenzie et al.,
2003) and in exercised GH-transgenic salmon
(Stevens et al., 1998)
compared with non-transgenic fish of the same mass. IDH provides NADPH for
lipid synthesis, suggesting a lower lipid synthesis in the liver of 7 month
old GH-transgenics. This result agrees with previous findings that
GH-treatment inhibited lipogenesis, and a reduced fat content was reported in
the carcasses of GH-treated (Sorensen et
al., 1996) and GH-transgenic
(Pursel et al., 1990) farm
animals. Moreover, Fauconneau et al.
(Fauconneau et al., 1997) also
reported a decrease in the size of adipose cells after GH treatment in rainbow
trout. The extra GH in these transgenics could also increase appetite and food
conversion and ultimately act on fish metabolism to reduce fat accumulation
(Fletcher et al., 2004).
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CONCLUSIONS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSReferences |
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Alfei, L., Onali, A., Spano, L., Colombari, P. T., Altavista, P. L. and DeVita, R. (1994). PCNA cyclin expression and Brdu uptake define proliferating myosatellite cells during hyperplastic muscle growth of fish (Cyprinus carpio L). Eur. J. Histochem. 38,151 -162.[Medline]
Allen, R. E. and Rankin, L. L. (1990). Regulation of satellite cells during skeletal muscle growth and development. Proc. Soc. Exp. Biol. Med. 194, 81-86.[Abstract]
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| |||||||||||||||||||||||||||
, MCs
extracted from 4 month old non-transgenic salmon; 
, MCs extracted from
7 month old non-transgenic salmon. Sample size equals 3 for transgenic and
non-transgenic salmon at 4 months (control age) and 5 for non-transgenic
salmon at 7 months (control size). Results are means ± s.d.; a two way
ANOVA was performed using the effects of days after plating
(P<0.001), presence of transgene (P<0.001) and the
interaction (P=0.0029); asterisk indicates significant differences
between transgenic and non-transgenic fish at 7 months (Bonferroni,
P<0.05).
