Hormonal control of swimbladder sonic muscle dimorphism in the Lusitanian toadfish Halobatrachus didactylus
Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
* Author for correspondence (e-mail: tmodesto{at}ualg.pt)
Accepted 2 July 2003
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Summary |
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Key words: muscle hypertrophy, androgen, castration, male dimorphism, toadfish, Halobatrachus didactylus
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Introduction |
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The sonic organ of batrachoidids is the swimbladder. Sound production
results from the contraction of paired sonic muscles attached to the walls of
the swimbladder, which cause a rapid variation in swimbladder volume and
internal pressure (Skoglund,
1961; Tower,
1908
). Sonic fibers have a number of morphological and biochemical
adaptations for rapid contraction speed: (1) an unusual radial morphology
[fibers are of polygonal shape and contain a core of sarcoplasm surrounded by
a contractile cylinder of alternating slender ribbons of sarcoplasmic
reticulum (SR) and myofibrils; Fawcett and
Revel, 1961
; Fine et al.,
1993
; Loesser et al.,
1997
], (2) an extremely well-developed SR forming about a third of
the fiber volume (Appelt et al.,
1991
; Franzini-Armstrong and
Nunzi, 1983
) and (3) the calcium transient is the fastest ever
recorded in a vertebrate muscle (Rome et al.,
1996
,
1999
). The sarcoplasm
immediately beneath the cell membrane and inside the core contains glycogen
granules and mitochondria, but, unlike most muscle fibers, mitochondria are
excluded from the contractile portion of the fiber. The functional design of a
radial arrangement of alternative ribbons of SR and myofibrils minimises the
distance and thus the transport time of calcium to the contractile proteins
and back to the SR (Fine et al.,
1993
).
The swimbladder and sonic muscles of toadfishes and midshipmen increase in
size throughout life and are larger in males than in females; this sexual
dimorphism appears to be related to the type of sound produced
(Brantley et al., 1993b;
Fine, 1975
;
Fine et al., 1990a
). Both
sonic muscle growth and enzyme activity have been shown to be androgen
sensitive (Pennypacker et al.,
1985
; Walsh et al.,
1989
). The mass of the swimbladder-sonic muscle complex is
increased by androgen implants in gonadectomised males and females of the
oyster toadfish Opsanus tau (Fine
and Pennypacker, 1986
) and in juvenile males and females and
sneaker/satellite males (type II males) of the plainfin midshipman
Porichthys notatus (Brantley and
Bass, 1991
; Brantley et al.,
1993a
). Additionally, androgen implants also influence fiber
morphology by increasing the area of sarcoplasm and thereby mitochondria
density (Brantley et al.,
1993a
).
In the Lusitanian toadfish, a significant increase in the sonic muscle mass
of type I males occurs during the breeding season, whereas females and type II
males do not show this increase (Modesto
and Canario, 2003). One hypothesis to explain this seasonal
hypertrophy is that elevated levels of 11-ketotestosterone (11KT) at the
beginning of the reproductive period modulate the growth of sonic muscle,
since, among reproductive males, 11KT but not testosterone (T) is elevated in
type I males compared with type II males
(Modesto and Canario, 2003
).
Considering the potency of 11KT at inducing secondary sexual characters in
other teleosts (Borg, 1994
),
this steroid is a strong candidate for generation and/or maintenance of sonic
muscle sexual dimorphism.
The present study investigated whether H. didactylus sonic muscle size and/or sonic muscle fiber morphology are indeed under androgenic control. Firstly, we have examined the morphological changes in sonic fiber structure underlying the seasonal changes in sonic mass of females, type I and type II males. Secondly, we have evaluated the responsiveness of sonic muscle to steroid treatment. Finally, we have analysed the long-term effect of castration on sonic muscle mass and on fiber structure.
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Materials and methods |
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Animals used in laboratory experiments were collected in Ria Formosa and kept in 1 m3 tanks in running seawater, at natural water temperature and photoperiod and fed three times a week on frozen squid.
Hormone implants
Synthetic steroids were purchased either from Steraloids (Newport, RI, USA)
or Sigma-Aldrich Chemical Co. (Madrid, Spain) and kept at 4°C in ethanol
stock solutions (1.5 µmol l-1). Steroids were administered
intraperitoneally as a liquid suspension (10%) in warm coconut oil
(Leatherland, 1985). As
coconut oil solidifies below 25°C, implants formed a single long mass
inside the body cavity and acted as a slow-release pellet.
Expt A - in vivo steroid release by implants
This experiment was carried out to determine the dynamics of steroid
release from the coconut oil implants. Type I males collected in May were
anesthetized with 2-phenoxyethanol (0.2 ml l-1; Sigma-Aldrich
Chemical Co.), tagged in the operculum with Presadon tags (Chevillot, France),
measured to the nearest mm to give total length (LT) and
weighed to the nearest 0.1 g to give total (MT) and
eviscerated mass (ME). Mean (± S.E.M.)
LT and ME of the fish used in the
experiment were 207±6 mm (range 181-241 mm) and 176.5±16.1 g
(range 132.7-262.1 g), respectively. Six fish received implants containing T.
The volume of implants was proportional to body mass and was administered
intraperitoneally at a steroid dose of 10 µg g-1 (group 1) or
100 µg g-1 (group 2). Three control fish received implants
without hormone (group 0). Blood samples (200 µl) were collected from the
caudal vein in heparinised syringes immediately before implantation and 0.5,
1, 2, 4, 7, 14, 21 and 35 days later. Plasma was separated by centrifugation
(7500 g, 5 min) and stored at -20°C until analysis.
Expt B - short-term effects of castration and steroid replacement on swimbladder mass and fiber morphometry
H. didactylus type I males were collected in May and acclimated to the laboratory for one week. They were then anesthetized, tagged, measured, weighed and a blood sample collected as described above. Mean values of LT and ME of the fish used in the experiment were 228±5 mm (range 172-302 mm) and 204.2±12.1 g (range 94.5-431.4 g),respectively.
Castration was carried out by placing fish on their dorsal side on a table and making an incision in the abdomen of approximately 3 cm. Testes and accessory glands were removed and weighed. Throughout the procedure fish were maintained immobilised by pumping a continuous flow of aerated water carrying anaesthetic via the mouth over the gills. The incision was sutured and an antiseptic (betadine; Mundipharma, Zurich, Switzerland) applied to the wound. As controls, some fish received the incision, and testes were exposed, handled and sutured (SHAM), while others were not operated on (INT). To directly test whether steroids can stimulate muscle hypertrophy or alterations in fiber morphology, castrated fish received coconut oil implants containing 100 µg g-1 of T, 11KT or 17ß-estradiol (E2) for a period of six weeks. A control group received steroid-free implants. Hence, the experimental groups were: intact (INT), sham-operated (SHAM), castrated (CAST), castrated plus control implants (CAST + C), castrated plus T implants (CAST + T), castrated plus 11KT implants (CAST + 11KT) and castrated plus E2 implants (CAST + E2). The number of fish per group is indicated in Table 1. Fish were allowed to recover from anesthesia in individual receptacles with aerated seawater before being transferred to separate experimental tanks for each group. No food was offered for the first two days after surgery. Two weeks after implantation, blood samples were collected from lightly anesthetized fish for steroid analysis. At the end of the experiment (six weeks after implantation), the fish were measured and weighed and blood collected as described above. The animals were sacrificed by spinal transection and the swimbladders removed, weighed and fixed for histological analysis.
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Expt C - long-term effects of castration on swimbladder mass and fiber morphometry
Adult H. didactylus were caught in December and surgically castrated as described above. Mean LT and ME values for the animals used in the experiment were 251±5 mm (range 197-340 mm) and 282.0±18.5 g (range 105.6-782.0 g),respectively. The experimental groups were: type I males intact, i.e. not operated and not castrated (MI INT), type I males castrated (MI CAST), type II males intact (MII INT), type II males castrated (MII CAST) and a group of intact females (F INT). The number of fish per group is indicated in Table 2. Fish were maintained in the experimental tanks for six months after castration. By the time of natural spawning in the field, June, all groups were sacrificed and sampled as described for experiment B. In experiments B and C, at autopsy the body cavity of castrated fish was inspected for testicular and/or accessory testicular gland remnants.
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Histology and histomorphometry
Swimbladders were fixed in Bouin's fluid for 48 h and then transferred into
70% ethanol. Sonic muscle fibers extend in a plane roughly perpendicular to
the rostrocaudal axis, aligned in concentric layers in a C-shaped
course. Fixed muscles were cut with a blade along the rostrocaudal axis in the
plane that cross-transected the muscle fibers to define the plane for
subsequent sectioning. The muscle slices were embedded in paraffin, sectioned
(5-7 µm) and stained with hematoxylin and eosin (H&E).
Muscle structure was analysed using the OPTIMAS 5.2 computerized image analysis system (BioScan, Inc., Edmonds, WA, USA). Mean areas of fiber components were derived from measurements of 50 randomly chosen cells in each swimbladder. Video images of the cells were digitized to generate cross-sectional areas of the muscle fibers (total fiber area), the myofibril-containing zone (myofibril area) and the peripheral sarcoplasm (sarcoplasm area) (see Fig. 1). In H. didactylus, the sarcoplasmic central core of myofibrils, which is present in adult fibers of other toadfishes, is nonexistent or barely visible by light microscopy and is therefore not measurable.
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Steroid assays
Sex steroids in plasma were measured by radioimmunoassays (RIAs) following
the methodology described in Scott and Canario
(1992); the origin and
cross-reactions for the T, 11KT and E2 antisera have been
previously reported by Scott et al.
(1984
), Kime and Manning
(1982
) and Guerreiro et al.
(2002
), respectively.
Intra-assay and inter-assay precision (coefficient of variation) were 7.5% and
12.4% for T, 8.2% and 11.6% for 11KT, and 8.0% and 8.8% for E2,
respectively. The limit of detection of assays was 100 pg ml-1 for
E2 and T and 160 pg ml-1 for 11KT.
Statistical analysis
All data are expressed as means ± S.E.M. Data (masses,
section areas and hormone concentrations) were first log-transformed before
statistical treatment to obtain normality and homogeneity of variance.
Swimbladder and sonic muscle masses and morphometric measurements of sonic
muscle fibers were compared between groups using analysis of covariance
(ANCOVA), with eviscerated body mass as covariate. Least squares means and
their standard errors adjusted for eviscerated body mass are reported. Plasma
steroid concentrations were compared by two-way analysis of variance (ANOVA).
Tukey's honest significant difference test was used for post-hoc
comparisons among means. Statistical significance for all tests was considered
at the 5% level. Pairwise comparisons of fiber characteristics between the
intact females and each of the experimental male groups in experiment C were
made using linear contrasts in ANCOVA. The software SPSS (version 9.0, SPSS
Inc., Chicago, IL, USA) was used to carry out the statistical analysis.
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Results |
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Total swimbladder mass (MTS), sonic muscle mass (MM) and swimbladder wall mass (MS) were linearly related to eviscerated fish mass (ME) for both type I males and females: MTS=0.2790+0.0202ME, r2=0.922; MTS=0.0744+0.0168ME, r2=0.895; MM=0.5340+0.0127ME, r2=0.839; MM=-0.0701+0.0126ME, r2=0.902; MS=0.1290+0.0035ME, r2=0.673; MS=0.0796+0.0033ME, r2=0.597, respectively, for males and females (P<0.001 in all cases). During the reproductive months, type I males had swimbladders and sonic muscles 25% and 30% heavier than females, respectively (MTS, 3.24±0.05 g and 2.59±0.05 g; MM, 2.67±0.09 g and 2.05 g; adjusted means for type I males and females, respectively; P<0.0001), but swimbladder wall masses were not significantly different between sexes (0.71 ± 0.03 g and 0.63 ± 0.05 g for type I males and females, respectively; P=0.136).
Sonic muscle fibers exhibited seasonal changes in morphology (Fig. 2). In winter, total fiber area of sonic muscle was significantly smaller in type I males than in females or type II males, but no significant differences were found during late spring/summer. Total fiber area in type I males was also significantly larger in late spring/summer compared with winter. In winter, the myofibril area was significantly smaller in type I males than in females or type II males. In late spring/summer, type I males still had a myofibril area significantly smaller than females but not than type II males. In winter, the sarcoplasm area was similar between females and the two types of males, but in spring/summer it showed a significant increase only in type I males. If mean sarcoplasm area is scaled to mean myofibril area, the resulting ratio (sarcoplasm area/myofibril area) for type I males in spring/summer far exceeds those of females but not of type II males. Altogether, morphometric measurements of sonic fibers showed that during the breeding season type I males had smaller myofibril contracting zones surrounded by larger areas of peripheral sarcoplasm compared with females, while type II males showed intermediate (but not significantly different) values of sarcoplasm area/myofibril area ratio between type I males and females.
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Experiment A - in vivo steroid release by
implants
Plasma T levels in the implanted animals showed a sharp increase within 0.5
days of implantation (Fig. 3).
In group 1 (10 µg g-1 implant), mean T plasma levels reached
89.02±6.40 ng ml-1, while group 2 (100 µg g-1
implant) reached significantly higher levels of 175.05±8.66 ng
ml-1. In both groups, a hyperbolic decay of T plasma levels
followed, and after a further two weeks T levels in group 1 and group 2 were
5.40±3.47 ng ml-1 and 24.00±8.76 ng ml-1,
respectively. At this time, plasma T levels in group 1 no longer differed
significantly from the control group (P=0.674). Five weeks after
implantation, group 2 still had significantly higher plasma T levels than
groups 1 and 0 (6.24±0.17 ng ml-1 vs
2.12±0.72 ng ml-1 and 2.64±1.13 ng ml-1,
respectively; P<0.05). Plasma T levels of control animals did not
change significantly over the duration of the experiment
(P=0.250).
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Experiment B - short-term effects of castration and steroid replacement on swimbladder mass and fiber morphometry
Castrated animals showed a reduction in T, 11KT and E2 levels after six weeks compared with initial values and with levels of intact (INT) and sham-operated (SHAM) animals (P<0.05; Fig. 4). Two weeks after implantation, T, 11KT and E2 levels were significantly higher in the respective hormone-implanted groups (P<0.05). In addition, E2 levels were elevated in the CAST + T group after 6 weeks (P<0.05), indicating some degree of extra-gonadal aromatization.
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Adjusted swimbladder masses of SHAM animals were not significantly different from those of INT animals, showing that surgery itself had no effect (P=0.410; Table 1). Additionally, CAST animals had an MTS similar to INT animals (P=0.200). Only E2-implanted castrated animals (CAST + E2) showed a significant reduction in MTS compared with control animals (CAST + C; P<0.05; Table 1).
Neither the incisions nor the castration had significant effects on any of the structural features of sonic fibers (P>0.100; not shown). The hormone treatment had no significant effect on total fiber area (P=0.471), myofibril area (P=0.627) or sarcoplasm area (P=0.059) but did have a significant effect on sarcoplasm area/myofibril area ratio (P<0.01; Fig. 5): E2-treated animals exhibited a sarcoplasm area/myofibril area ratio significantly lower than that of the control (CAST + C; P<0.05).
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Experiment C - long-term effects of castration on swimbladder mass and fiber morphometry
In control groups of both male morphs, plasma levels of androgens increased significantly from the beginning of the experiment, reflecting normal testicular recrudescence. By contrast, plasma levels of castrated groups decreased significantly during the experimental period (P<0.05; Fig. 6).
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Six months after castration, there were significant differences in MTS between the control and castrated groups for both types of males (P<0.01; Table 2). Castrated fish had a lower MTS than non-castrated fish. The swimbladders of both morphs responded similarly to castration, both in mass (morph x treatment, P=0.115) and in morphology of sonic muscle fibers (morph x treatment, P>0.9 for any sonic fiber characteristics).
Castration increased total fiber area (marginally significant, P=0.048) and myofibril area (P<0.001) in both morphs but had no effect on sarcoplasm area (P=0.836). This was reflected in the ratio of sarcoplasm area/myofibril area, which showed a significant decrease in castrated groups (P<0.001; Fig. 7).
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Castrated type I males were similar to females in sarcoplasm area (P=0.989) and sarcoplasm area/myofibril area ratio (P=0.477), while castrated type II males were similar to females in all of the fiber characteristics (total fiber area, P=0.964; myofibril area, P=0.666; sarcoplasm area, P=0.649; sarcoplasm area/myofibril area ratio, P=0.975).
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Discussion |
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During the breeding season, the general hypertrophy of sonic muscle mass in
H. didactylus males was accompanied by structural modifications in
muscle fibers. Males had thinner myofibrils and more sarcoplasm and,
therefore, a bigger sarcoplasm area/myofibril area ratio than females, which
is similar to what has been observed in male weakfish (Cynoscion
regalis; Connaughton et al.,
1997) and the toadfish O. tau
(Fine et al., 1990a
). For the
toadfish, Fine et al. (1990a
)
hypothesized that the thinner myofibers in males are an adaptation to the
increased speed and fatigue resistance necessary for production of the
boatwhistle mating call, since the small size and concomitant large
surface-to-volume ratio would facilitate rapid fluxes of glucose, oxygen,
lactic acid and CO2. Additionally, enlargement of the sarcoplasm
area permits larger mitochondria content around the myofibril zone, essential
to the energetic demands of boatwhistles. Appelt et al.
(1991
) found that sonic muscles
in males have approximately three times as many mitochondria as those in
females. Electron microscopy has shown that in O. tau the larger
fibers develop fragments of a contractile cylinder separated by channels of
expanded sarcoplasmic reticulum (containing glycogen granules and
mitochondria) that will form new, smaller and, therefore, more energy
efficient fibers (Fine et al.,
1993
). In H. didactylus, although we did not measure the
number of muscle fibers, the enlargement of the sonic mass in type I males
during the breeding season suggests that hyperplasia of sonic fibers occurs
and, together with alterations of sonic fiber morphology, may explain the
ability to produce boatwhistle calls (dos
Santos et al., 2000
). In winter, when long calls are not used in
mating behavior, sarcoplasm areas of fibers from females and type I male were
not significantly different, possibly reflecting similar energetic and
mechanical demands to produce mainly agonistic short calls by both sexes.
Comparison of the characteristics of the swimbladder and sonic fibers among batrachoidids shows that the midshipman P. notatus exhibits a pronounced swimbladder dimorphism compared with the two toadfishes (Table 3). Nesting midshipman (type I) males have a much bigger sonic muscle mass, which is related to the elevated number of larger fibers, with larger myofibrils and larger peripheral and central zones of sarcoplasm than females (or sneaker/satellite type II males).
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Additionally, inter- and intrasexual dimorphism of the peripheral sonic
organs of P. notatus is also evident in the morphophysiological
characteristics of the neuronal pathway controlling sonic muscle: type I males
have larger cell bodies, dendrites, axons and terminal junctions between
nerves and muscles, and pacemaker neurons fire at higher frequencies than in
females or type II males (Bass and
Marchaterre, 1989; Bass,
1996
; Bass and Andersen,
1991
; Fluet and Bass,
1990
). In the toadfish O. tau, although no morphological
differences in body size and sonic muscle characteristics are evident among
males, as occurs in P. notatus, sonic motoneuron histology has showed
that males can be separated into two groups - one with large motoneurons and
the other with small motoneurons - but both morphs have similar dendrite
diameter and neuron number. Females show morphological neuron characteristics
similar to those of males with small motoneurons
(Fine, 1997
).
In contrast to P. notatus, where type II males are much smaller
than type I males, H. didactylus type II males are not markedly
different in size from type I males or females. In H. didactylus,
there was a partial overlap in body sizes of females, type I males and type II
males, although only type I males were found larger than 220 mm
(Modesto and Canario, 2003).
The moderate inter- and intrasexual dimorphism in body size of H.
didactylus also applies to swimbladder masses and to features of sonic
fibers and resembles that of the toadfish O. tau. During the breeding
season (late spring/summer), type II male swimbladders were significantly
heavier than those of females but were lighter than those of type I males,
while both myofibril area and the ratio of sarcoplasm area/myofibril area of
type II male fibers was intermediate, although not significantly different,
between those of type I males and females. How these differences correlate
with distinct characteristics of the neuronal circuitry, capacities in sound
production and reproductive behavior still needs to be investigated.
In H. didactylus, six months but not six weeks of castration
reduced swimbladder masses and decreased sarcoplasm area/myofibril area ratio.
Furthermore, males castrated for six months showed morphological
characteristics of fibers similar to those of intact females. Since castration
of H. didactylus was performed in December, before the period of
natural increase of sonic mass, results obtained after six months (experiment
C) may indicate that testicular factors are required to initiate seasonal
sonic muscle hypertrophy. However, these factors do not seem to be necessary
to maintain sonic hypertrophy for short periods, since six weeks after
castration (experiment B), during the period of sonic hypertrophy, no
alterations in sonic mass or fiber morphology were observed. Similar negative
results for swimbladder masses of short-term (3-4 weeks)castration have been
obtained for O. tau (Fine and
Pennypacker, 1986). Results obtained for short-term castration are
corroborated by the fact that swimbladder masses of type I males of H.
didactylus in nature peaked in June but stayed significantly higher until
November, when gonadosomatic index and plasma androgen levels fell to the
lowest levels (Modesto and Canario,
2003
). So, it is possible that seasonal hypertrophy experienced by
the sonic muscles may be triggered by an increase in plasma androgen levels in
spring and by the progressive workload of sonic muscles to produce mating
calls. By contrast, the slight decrease in sonic mass during the months after
spawning (mid- to late summer) may be the result of a slow decrease in the use
of sonic muscles and by the low levels of androgens.
Androgen implants (T and 11KT) did not affect sonic muscle mass in H.
didactylus. However, E2 caused a significant decrease in
swimbladder mass and a reduction in the sarcoplasm area/myofibril area ratio,
which possibly resulted from a slight simultaneous decrease in sarcoplasm area
and an increase in myofibril area, both features characteristic of females.
These effects of E2 implantation were similar to those induced by
the long-term castration and resemble the inhibitory effect in the development
of type IIA skeletal fibers (characteristic of swimbladder muscles;
Fine and Pennypacker, 1988)
induced by E2 in rats (Koboni
and Yamamuro, 1989
; Suzuki and
Yamamuro, 1985
). Walsh et al.
(1995
) cited unpublished
results by Mommsen and Bass in which treatment of adult type I males of P.
notatus with E2 for a few weeks resulted in significant
decreases in the overall mass of sonic muscle.
The effects of androgens either on sonic mass or on muscle fiber structure
of H. didactylus were not as pronounced as reported in other
teleosts. For example, in P. notatus, androgen (T and 11KT)
implantation for nine weeks markedly increased the relative sonic muscle size
in juvenile males, juvenile females and type II males, whereas E2
and cholesterol had no effect. The principal androgen effect on fiber
structure was an increase in the area of mitochondria-filled sarcoplasm, and
thus the sarcoplasm area/myofibril area ratio increased by 1.4-2-fold in the
androgen-treated groups (Brantley et al.,
1993a). In the toadfish O. tau, although no seasonal
pattern was found for changes in total swimbladder mass, sonic muscle mass or
sonic motor nucleus neuron size (Johnson
et al., 2000
), sonic muscle was stimulated to grow by androgen (T
and dihydrotestosterone) implants for 3-4 weeks both in males and in females,
while E2 implants only caused a significant increase in females but
not in males (Fine and Pennypacker,
1986
). Sonic muscle enzyme activity was also increased by
androgens (Pennypacker et al.,
1985
) but had no effect on sonic motor nucleus neuron size, and
these neurons did not concentrate androgens (T and dihydrotestosterone) or
E2 (Fine et al.,
1982
,
1990b
,
1996
). In C. regalis,
a sciaenid species in which males have extrinsic sonic muscles, sonic muscle
mass of T-implanted groups increased 2.5-fold over a period of three weeks
compared with sham-implanted groups. In T-implanted groups, the
myofibril-contracting zone of sonic fibers was significantly greater than
those of the sham-implanted and time-zero groups, but little increase in
sarcoplasmic area was noted (Connaughton
and Taylor, 1995
).
The failure of the sonic muscle in H. didactylus to respond to androgen could have been a consequence of several factors: (1) an inadequate dose of androgen and/or duration of the experiment, (2) the muscle already being at its maximum stimulation when implants were applied or (3) in this species other factors rather than androgens being responsible for sonic muscle hypertrophy and hyperplasia.
In the present study, we have tested the effect of sex steroids over a
period of six weeks after a single implantation of 100 µg g-1.
This dose was similar to that used in O. tau
(Fine and Pennypacker, 1986)
and in C. regalis (Connaughton
and Taylor, 1995
) over a period of 3-4 weeks, which induced
significant alterations in sonic muscle mass and fiber morphology.
Additionally, during our study, plasma levels of T and 11KT were always higher
(experiment A) than those found in animals collected in nature during the same
period (Modesto and Canario,
2003
). So, it seems unlikely that dose/duration factors by
themselves could explain the inefficiency of androgens to induce alterations
in sonic muscles.
In nature, swimbladder masses of type I males start to increase in March
and peak in June when plasma T and 11KT reach their highest values
(Modesto and Canario, 2003).
It is therefore possible that when steroid implants were applied (mid-May),
the sonic muscle was already fully stimulated due to a period (from January)
of continuous relatively elevated levels of androgens (particularly 11KT;
Modesto and Canario, 2003
) and
of a progressive workload of sonic muscles to produce mating calls. In
mammals, the intake of androgens and concomitant strength-training exercise
have a synergetic effect and can induce an increase in muscle protein
synthesis (Lamb, 1975
), which
is reflected in an increase in cross-sectional area of muscle fibers and
formation of new muscle fibers (Alen et
al., 1984
; Kadi et al.,
1999
).
In P. notatus, androgen implants markedly induced the increase of
sonic muscle size and the muscle cell phenotype typical of type I males in
juvenile males but not in a similar way in juvenile females or type II males.
This suggests that sexually immature males have a unique sensitivity to
androgen-induced changes in sonic muscle mass and architecture, but type II
males and females have either lost this sensitivity at some point in their
life history or, in fact, never possessed it
(Brantley and Bass, 1991;
Brantley et al., 1993a
). This
difference in responsiveness of type I and type II males to androgens
emphasizes the existence of different developmental trajectories between males
(Bass, 1996
). In some teleosts,
a critical period exists during which organizational changes in morphology can
be elicited by steroid treatment or gonadectomy. Following this period, full
reversal cannot be totally accomplished through either method
(Adkins-Regan, 1981
). Since we
have used adult mature type I males for steroid treatment, it is possible that
if a critical period (age- and/or season-dependent hormone effects) exists in
H. didactylus for the expression of sonic muscles it had already
passed for these specimens prior to the time of experiments.
Moreover, muscle growth in vertebrates is often attributed to other factors
such as growth hormone and insulin-like growth factors
(Florini, 1987;
Florini et al., 1996
), and it
cannot be ruled out that non-steroidal mechanisms (or other non-androgen
steroids) could be involved in the development of sonic muscles in H.
didactylus. Future studies to detect androgen/estrogen receptors in sonic
muscles of H. didactylus would be important, since the presence of
such receptors should reflect tissue hormone sensitivity.
In conclusion, although factors derived from the testes appear to be required for sonic muscle seasonal hypertrophy and hyperplasia, the involvement of androgens, as demonstrated for O. tau and P. notatus, was not found in H. didactylus, possibly because of a previous endogenous stimulation of adult type I males. Further studies testing specific sensitivity to inductive effects of androgens during winter and with juveniles and females are required to clarify this aspect and to understand swimbladder sonic muscle ontogeny.
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