Acute Effects of Dihydrotestosterone on the Contractile Kinetics of Isolated

Bufo marinus Gastrocnemius during Low Frequency Fatigue

Claudia Gemes, Michael Huang, Jancy Johnson, Seungkyum P. Kim, Ben McDermott, Peter Nguyen Tran, Brooke Phillips and Joyce
Wang.
Department of Physiology, The University of Melbourne, Victoria, Australia

Key Points summary:

Dihydrotestosterone (DHT) has both acute and non-genomic effects targeting muscle contractility.
Low-frequency fatigue (LFF) reduces muscle contractility and force production for prolonged periods
of time.
In the present study, we indicate that DHT has no significant effect in the reversal of depressed force
production and delayed skeletal muscle recovery associated with LFF.
Our data indicates that DHT has limited therapeutic use and medical applications in the symptomatic
relief of muscle weakness associated with neuromuscular disorders.

Abstract:
Low-frequency fatigue has been a major component of whole muscle fatigue associated with repetitive, submaximal
exertion affecting the calcium component of the excitation-contraction coupling of skeletal muscles. Using dissected whole
muscle gastrocnemius of male bufo marinus, the aim of this study was to therefore investigate the acute, non-genomic
effects of dihydrotestosterone (DHT) in overcoming fatigue. Via the use of a Watson Victor LTD force transducer, LabChart
software (ADInstruments, Dunedin, NZ) and PowerLab 4/20 hardware, fatigue was electrically evoked via the
administration of a constant-frequency train of single pulse stimulations at 20Hz frequency, 49.95 ms duration and at 150300mA until tetanic force reduced by >50% of the pre-fatigue maximal twitch force. Despite the depression in force
production, we found that DHT had no role in the sarcoplasmic reticular releases of calcium, nor in its sequestration. This
was evident in a comparison of the maximal forces produced in nerve-muscle preparations subjected to DHT (10948g)
and those to saline (13164g). Furthermore, we found that DHT did not significantly delay the onset of fatigue (Control:
456.5152.2s; Flutamide: 421.2147.8s; DHT: 522.2 204.0s; DHT+Flutamide: 500.0242.7s) and additionally had no
bearing on twitch force recovery (Control: 7814%; Flutamide: 7218%; DHT: 8014%; DHT+Flutamide: 7211%) in a
measurement of post and pre-fatigue twitch forces between each group. Mechanistically, we were also unable to support
the independent function of DHT from the androgen-receptor pathway as there was no significant difference evident in the
contractile kinetics found between the function of DHT in presence and absence of flutamide.
Abbreviations: AR, Androgen Receptor; DHT, Dihydrotestosterone; EDL, Extensor Digitorum Longus; EGFR, Epidermal
Growth Factor Receptor; EC coupling, Excitation-Contraction Coupling; FCR, Flexor Carpi Radialis; FES; Functional Electrical
Stimulation; RT, Half-Relaxation Time; HFF, High-Frequency Fatigue; IP3, Inositol Triphosphate; LFF, Low-Frequency
Fatigue; MAPK pathway, mitogen-activated protein kinase pathway; PCD, Postcontractile Depression; SR, Sarcoplasmic
Reticulum; SERCA, sarco-endoplasmic Reticulum Calcium ATPase; TA, Tibialis Anterior

Introduction
Existing literature has insofar suggested that
Dihydrotestosterone (DHT) may have a significant role
in influencing the contractile kinetics of skeletal
muscles (Melichna et al., 1972) due to either an acute
mechanism affecting the actin-myosin interaction
(Saboridom, Molano & Megias, 1991; Hamdi &
Mutungi, 2010), or the mediation of its effects via a
genomic pathway (Evans & Ivy, 1982). This could hold
potential therapeutic and supplementary purposes for
patients who suffer from neuromuscular disorders or
spinal cord injury. In addition to physiotherapy, use of
DHT could possibly aid in the rehabilitation of patients
who experience muscle weakness, hereby targeting
diminished muscle function in a way that could
facilitate eventual rehabilitation (Green, Robinson &
Wallis, 2014). Likewise, this is a phenomena often
characterised by the reversible loss of muscle function
efficacy, described by previous literature as fatigue
(Allen, Lamb & Westerblad, 2008). Having both a
central and a peripheral component, peripheral
fatigue concerning the effective exhaustion of muscle
fibres independent of the central nervous system has
two further components understood as low-frequency
fatigue (LFF) and high-frequency fatigue (HFF). This
describes either rapid or delayed force recovery
observed at either low or high frequency stimulations
respectively (Edwards et al., 1977) and may be
mediated through neuromuscular electrical

stimulations or chronic muscle exertion resulting in a

reduction in the ability to produce force during
exercise (Gandevia, Allen & McKenzie, 1995; Roja et
al., 2006).
In light of the importance of LFF and its rehabilitative role
in functional electrical stimulation (FES), daily activities
involving voluntary skeletal muscle activation also very
rarely exceeded 30Hz, typically considered as a lowfrequency in terms of motor unit discharge (BiglandRitchie, Jones & Woods, 1979). This has therefore made
LFF a good candidate for further investigation as opposed
to that of HFF given its correlation to rehabilitation, and
commonality of LFF-related incidents during everyday
situations. In addition, the mechanistic effects of LFF has
an overall overlap to that of DHTs mechanistic pathway,
which targets the calcium transient component of actinmyosin interaction as suggested by previous literature
(Jones, 1981; Westerblad, Duty & Allen, 1993; Allen et al.,
1995; Jones, 1996; Bruton et al., 2008; Hamdi & Mutungi,
2010; Oliver et al., 2013). In one study, Baptista et al.
(2009) administered electrical stimulations at the
frequencies of 20Hz and 100Hz to induce repeated short
tetani in human skeletal muscles (Ratkevicius et al., 1998;
Skurvydas et al., 2000; Allen, Lamb & Westerblad, 2008)
observing that the effects of LFF at 20Hz was more
pronounced and in greater alignment with the
observation of significantly prolonged and reduced

forces, than those observed at 100Hz.

frequency stimulations. Measurement of intracellular

changes in calcium concentrations provided promising
These findings have also been reported upon by previous results for further recommended research, suggesting
other experiments involving both human and animal
that potentially, this deficiency in calcium transient
skeletal muscle preparations, noting that, at lower
component of EC coupling induced in the fatigued state
frequencies, a greater sensitivity in the force-generating
could be reversed via application of DHT.
capacity of skeletal muscles was evident to even slight
Therefore, working off the mechanism proposed by Oliver
changes to intracellular calcium concentrations (Jones,
et al. (2013) and Hamdi & Mutungi (2011), we suspect
1996; Westerblad et al., 2000; Rijkelijkhuzien et al., 2003;
that DHT mediates its effects via an interaction with a
Keeton & Binder-Macleod, 2006; Bruton et al., 2008;
DHT sensitive receptor independent to that of the
Watanabe et al., 2014). In light of these findings, our use
Androgen receptor (AR) (Steinsapir et al., 1991;
of 20Hz frequency as part of our fatiguing protocol is
Gorczynska & Handelsman, 1995) at the sarcolemma
closely modelled to that described by Edwards et al.
resulting in the activation of a series of secondary
(1977), involving a similar use of a constant-frequency
messengers that increases sarcoplasmic reticular (SR)
train to induce a tetanic response following submaximal
calcium release. This would subsequently effect a
activity. This is in the hopes of observing the effects of
measurable change in force production via EC coupling
DHT in targeting and reversing the calcium component
measured via our use of the Watson-Victor Ltd force
adversely affected by the induction of LFF.
transducer. Our study aims to use an androgen receptor
inhibitor, Flutamide, in order to determine if whether or
We expect to measure the consequences of this action
not the DHT-mediated elevation in intracellular calcium
via the subsequent contractile forces produced by the
releases occurs via a different pathway using the whole
nerve-muscle preparation in order to yield novel results.
gastrocnemius muscle of b. marinus as a model to analyse
Additionally, our use of the amphibian model via in vitro
the acute effects of DHT in the presence of LFF. We
dissection of whole muscle gastrocnemius of male b.
hypothesise that the administration of DHT will have a
marinus may also provide novel insight into an area of
restorative role in twitch force production following
study previously dominated by the use of rat, mice or
fatigue. In a similar vein to other studies measuring the
muscle fibre models. Westerblad, Lnnergren & Allen
half-relaxation time ( RT) (Peters & Aulner, 2000; Clark
(1997) explored this avenue and suggested that
& Peters, 2006; Kampes & Peters, 2013), we will also
alterations in the calcium handling and the cross-bridging measure the time required for force production to drop
component of skeletal muscle contraction of Xenopus
beneath a 50% threshold of the initial maximal baseline
frog had significant contributions to delaying force
peak, hypothesising that DHT will work to delay fatigue
recovery in fatigue-induced slow and fast-twitch muscle
onset as well.
fibres. This link between the onset of fatigue and calcium
transient was furthermore explored in the amphibian
model by Usher-Smith et al. (2006), highlighting the
integral role of intracellular calcium concentrations in the
generation of force in skeletal muscle, with the
subsequent reduction in calcium release having obvious
downstream consequences for Excitation-Contraction
(EC) coupling following repeated tetanic stimulations
(Kabbara & Allen, 1999).
Although, Watanabe et al. (2014) used a rat model, they
also examined the relationship between the inherent
effects of LFF affecting calcium release and force
production in the context of skeletal muscle recovery,
providing results which suggested that, in a comparison
of fatiguing protocols using 20 Hz and 50Hz as low and
high frequency stimulations respectively (Edwards et al.,
1977; Jones, 1996; Chin & Allen, 1996), that the loss of
tetanic force was significantly more profound with lowfrequency stimulations compared to that of high-

Methods
Animals:
The experimental protocol was designed and
performed in accordance with The University of
Melbourne Animal Ethics Committee. The experiment
involved the use of 32 adult male b. marinus toads
(weight: 0.6619g0.1326g) sourced from Queensland,
Australia, that were subsequently housed
appropriately before being injected with their
respective pre-treatment as outlined in the
experimental procedure below. The toads were then
pithed under administration of general anaesthetic 40
minutes prior to the commencement of the
experimental protocol. All procedures were
performed under strict adherence to the guidelines
outlined by the ethics committee. The toads were
then dissected in order to isolate the gastrocnemius
muscle with the exposure of the intact sciatic nerve
ensured. All nerve-muscle preparations involved the
use of a hook through the ligamentum patella and a
suture threaded at the distal end of the skinned
gastrocnemius muscle for later attachment to an 800g
weight during the preparation of the force-transducer
apparatus. Following the fatiguing protocol, all nervemuscle preparations were subsequently excised of all
nerve, ligament and blood vessels and weighed
accordingly.
Solutions:
Preparation of organ baths at 25C room temperature
involved the formulation of Ringers solution of
composition: NaCl, 7.2; KCI 0.28; CaCl2 0.13; NaHCO3
0.2g/l. The injection concentration of 10ng/mL of
4,5-dihydrotestosterone (DHT) into living tissue was

loosely derived, and adjusted with an ethanol vehicle,

from the physiologically circulating concentrations of
DHT in wild type male cane toads sampled during the
breeding season of Queensland, Australia (Harvey et
al., 1997; McCoy et al., 2008). The use of androgen
receptor inhibitor, flutamide (2-Methyl-N-(4-nitro-3
[trifluoromethyl] sourced from Sigma-Aldrich, Castle
Hill, NSW, Australia) was also used with an ethanol
vehicle. Briefly, a total of 32 dissected gastrocnemius
muscles were divided into four groups each consisting
of eight nerve-muscle preparations subjected to either
the pre-treatments of saline (Group 1), flutamide
(Group 2), DHT (Group 3) and DHT+Flutamide (Group
4). Group 1 (n=7) was subjected to a 200L injection
of 10% ethanol solution in saline; Group 2 (n=7) was
subjected to a 200L injection of 10% ethanol solution
in saline with 3g/mL of flutamide alone; Group 3
(n=8) was subjected to a 200L injection of 10%
ethanol solution in saline solution with 10ng/mL of
DHT alone; and Group 4 (n=8) was subjected to a
200L injection of 10% ethanol solution in saline with
a combination of 3g/mL flutamide and 10ng/mL
DHT. Nerve-muscle preparations that were excluded
from further analyses were non-functioning due to
damage to the sciatic nerve.
Setup of force transducer and measurements:
The nerve-muscle preparation was mounted to a
Watson Victor LTD force transducer calibrated with an
800g weight. During the experimental protocol, a
passive tension of 20g was employed throughout. All
measurements of force and time were facilitated via
the use of PowerLab 4/20 Hardware and LabChart
software (ADInstruments, Dunedin, NZ).

Stimulation and fatiguing protocol:

Facilitated via the use of a Watson Victor LTD force
transducer that measured active tension in grams, one
supramaximal single pulse twitch at 500mA, 100Hz
frequency was delivered to the nerve-muscle
preparation in order to pre-potentiate the muscle and
ensure the function of the sciatic nerve (Belanger,
McComas & Elder, 1983). In accordance with previous
literature (Edwards et al., 1977; Jones, 1996; Allen,
Lamb & Westerblad, 2007; Bruton et al., 2008), our
fatiguing protocol focused upon the induction of LowFrequency Fatigue (LFF) via the administration of
single pulse stimulations at 20Hz frequency delivered
in a constant frequency train of 1 second intervals, at
the optimal current amplitude (mA) with a short
pulse duration of 49.95ms (Allen, Lamb & Westerblad,
2007) utilised in accordance with the technical
limitations of the operating equipment and software.
The optimal stimulation amplitude (mA) of the nervemuscle preparation was determined via a series of
single pulse stimulations at 20Hz, 49.95ms with a 6
second resting gap in between each stimuli
administered at 10mA, 20mA, 30mA, 50mA, 75mA,
100mA, 125mA, 150mA, 200mA, 250mA and 300mA
(refer to table 1). Subsequent to this, the nervemuscle preparation was allowed a 5 minutes resting
period which was then followed by its subjection to up
to three single pulse stimulations at 20Hz, 49.95ms at
the optimal stimulation amplitude previously
determined. This was administered in order to
determine the pre-fatigue, baseline amplitude that
would be later used to calculate the percentage of
force recovery following the fatiguing protocol. The
fatiguing protocol was then immediately commenced
and terminated once the tetanic force dropped
beneath 50% of the initial pre-fatigue amplitude,
indicative of skeletal muscle fatigue (Fitts, 1994;
Gandevia, Allen & McKenzie, 1995; Jones, 1996; Allen,
Lamb & Westerblad, 2007; Baptista et al., 2009).
Following the fatiguing protocol, a 5 minutes resting
period (Keller et al., 2011) was then allowed before
the administration of up to three single pulse
stimulations at the aforementioned values. This would
function as our recovery amplitude that would then
be compared to the baseline amplitude that would, in
addition to recording the time to fatigue for the
nerve-muscle preparation, form the basis for our
analyses.
Statistics:
All statistical analyses were performed via the use of
SPSS 20.0 (IBM Inc., Armonk, USA). A comparison of
Group differences between Group 1 relative to Group

2 and Group 3, and Group 2 relative to Group 4 was

conducted for maximal force production, muscle
weight, time to fatigue, force recovery and percentage
recovery. Statistical significance was determined using
a one-way analysis of variance. The difference
between the baseline maximal force generated and
the recovery force peaks subsequent to the fatiguing
protocol within each Group for each nerve-muscle
preparation was also determined using a paired
sample t-test. P value for significance was set at 0.05.
Data are presented as meansSD. Exclusion of nervemuscle preparations occurred in the event that force
production was undetectable, force recovery
exceeded 100% or data was greater than 3 SD away
from the mean.

Results

Weight of gastrocnemius muscle. Overall, the nervemuscle preparations subjected to the saline treatment
(Group 1) showed a relatively similar mean mass
compared to the preparations subjected to flutamide
(Group 2), DHT (Group 3) and DHT & flutamide (Group
4) treatments (0.62120.1288g, 0.65930.1545g,
0.69300.1255g and 0.66970.1486g respectively)
with no significant differences reported across the
four groups (P>>0.05). Significance for Group 2 and
Group 3 were reported relative to that of Group 1,
whereas significance for Group 4 was reported
relative to that of Group 2 (fig. 1). There were no
outliers reported in terms of each individually
weighed gastrocnemius muscle.

stimulation of the sciatic nerve prior to the fatiguing

protocol was measured in grams and was then
standardized individually by dividing it by the
individual mass of the gastrocnemius muscle. There
were no significant differences (P>>0.05) between the
nerve-muscle preparations of Group 1 to Group 2 and
Group 3 (13164g, 11654g and 10948g respectively)
and Group 2 relative to Group 4 which reported a
similar force production amplitude of 13174g (fig. 2).
Notably for Group 2 however, one nerve-muscle
preparation (preparation 8) had a significant outlier of
404g reported relative to preparations 9, 10, 11, 12,
13, 14 and 15 (142g, 121g, 75g, 111g, 21g, 180g and
160g respectively) and had the effect of positively
skewing the data set. This result was therefore
excluded as it was up to three standard deviations
away from the mean.
A comparison of the pre-fatigue and post-fatigue
twitch force. The baseline maximal twitch force
recorded in grams of the nerve-muscle preparations
were similar between Group 1, Group 2, Group 3 and
Group 4 (13164g, 11654g, 10948g and 13174g
respectively) (fig. 2). Post-fatigue twitch force was
measured in grams (Group 1: 10253g; Group 2:
7932g; Group 3:8535g and Group 4: 9552g)
subsequent to the fatiguing protocol involving the use
of single pulse stimulations at 20Hz frequency,
49.95ms duration at the optimal current (mA) (refer
to table 1) at one-second intervals (fig. 3). Significance
was determined via a comparison of the two
aforementioned values (fig. 6), with results of each
nerve-muscle preparation compared against itself
within each group. Results reveal that even after the
recovery time frame of 5 minutes, the nerve-muscle
preparations of all four groups showed a significant
decrease in the amplitude of post-fatigue twitch force
recorded relative to that of the baseline pre-fatigue
twitch force (P=0.0243, P=0.0420, P=0.0131 and
P=0.0145 for Group 1, Group 2, Group 3 and Group 4
respectively).

Does DHT increase the maximal twitch force

produced? In determination of significance, all data
recorded for the force produced via electrode

Does DHT have an effect on twitch force recovery? In

light of the significant decrease in twitch force
production following the fatiguing protocol, we were
interested in examining the effects of DHT on twitch
force recovery. Therefore results were analysed
between groups. Significance in terms of the
percentage of force recovery was indicative of the
post-fatigue force amplitudes (Group 1: 10253g;
Group 2: 7932g; Group 3: 8535g; Group 4: 9552g)
as a fraction of the pre-fatigue force amplitudes
standardized to the individual mass of each muscle in

grams. The subsequent decay in force production

following fatigue is measured relative from Group 1 to
Group 2 and Group 3 (7814%, 7218% and 8014%
respectively) and Group 2 relative to Group 4 which
reported an overall percentage recovery of 7211%
for Group 4 (fig. 4). There was no significant difference
(P>>0.05) evident across all four groups. Most
notably, one nerve-muscle preparation (preparation
26) of Group 4 was considered to be a non-significant
outlier relative to preparations 24, 25, 27, 28, 29, 30
and 31 (68%, 75%, 75%, 64%, 71%, 57% and 72%
respectively), recording 96% force recovery. The result
was nonetheless included within the data set as it was
within two standard deviations.

dropped beneath at least 50% of the original prefatigue amplitude, with the subsequent data treated
as a fraction over the individual muscle mass recorded
in grams (fig. 5). Significance (P>>0.05) was
determined via a comparison of the nerve-muscle
preparations of Group 1 to Group 2 and Group 3
(456.5152.2 sec, 421.2147.8 sec and 522.2204.0
sec respectively) with a comparison of Group 2 to
Group 4 (500.0242.8 seconds) used as a basis for
significance. However, DHT showed no significant
difference relative to both Group 1 and Group 2. Large
variations in the data set, particularly for nervemuscle preparation 17 (823.2 seconds) for Group 3
relative to preparations 16, 18, 19, 20, 21, 22 and 23
(753.2 sec, 708.2 sec, 349.3 sec, 314.6 sec, 377.1 sec,
431.6 sec and 420.3 sec respectively) was evident. A
notably large but non-significant variation was also
evident in the results presented by preparation 31
(865.3 sec) relative to preparations 24, 25, 26, 27, 28,
29 and 30 for Group 4 (449.3 sec, 232.1 sec, 543.5 sec,
863.5 sec, 383.4 sec, 338.6 sec and 324.0 sec
respectively).

Discussion

Does DHT have an effect on fatigue resistance? Time

to fatigue was measured in seconds (sec) at the onset
of the fatiguing protocol until force production

Our experiment intended to investigate the novel

effects of dihydrotestosterone (DHT) on the
contractile kinetics of the gastrocnemius muscle of
male b. marinus within the context of overcoming
fatigue. Although previous literature had examined
the effects of low-frequency fatigue (LFF) in isolation
(Edwards et al., 1977; Westerblad, Duty & Allen, 1993;
Jones, 1996), the interaction between the impaired
sarcoplasmic reticular (SR) releases of intracellular
calcium associated with LFF, against the DHT-related
increases in intracellular calcium concentrations had
not yet been examined (Foradori et al., 2007; Oliver et
al. 2013). Nonetheless, our set use of 20Hz lowfrequency during the administration of single pulse
stimulations in a constant-frequency train induced a
reduction in tetanic force production by >50% of the
maximal baseline(Jones, 1996; Allen, Lamb &
Westerblad, 2008; Keller et al., 2011; Green, Robinson
& Wallis, 2014). This was used as a measure of fatigue
onset in accordance with fulfilling our aim. This
allowed us to examine the effects of DHT (Group 3) in
delaying and enhancing the fatigability and twitch
force recovery of the gastrocnemius respectivelyrelative to that of the saline controls (Group 1) and
within the presence of the androgen receptor (AR)
inhibitor, Flutamide (Group 2 and Group 4). All results
were standardized according to the individual mass of
the gastrocnemius (fig. 1).
Overall, our results were unexpected and indicated
that 10ng/mL injections of DHT had no significant

effect in delaying the onset of fatigue within the

dissected gastrocnemius, nor did it play much of a role
in influencing the contractile kinetics of the nervemuscle preparation. This did not support our
hypothesis as baseline maximal twitch force showed
no significant differences between groups subjected
to the saline pre-treatment (Group 1) and groups
subjected to the DHT pre-treatments (Group 3 and
Group 4), with no significant changes suggesting any
form of an interaction between DHT and flutamide in
terms of androgen-sensitive receptors. Nonetheless,
our data suggests that the interaction between DHT,
flutamide and AR is yet unclear, and that the
independence of DHT from the AR pathway could not
be dismissed. It was observed however, that there
was a significant reduction in force production
following the recovery period (P=0.0243, P=0.0420,
P=0.0131, P=0.0145 for Group 1, Group 2, Group 3
and Group 4 respectively) that was overall suggestive
of a prolonged depression in force production
(Westerblad & Lnnergren, 1986). This indicated at
least some change in calcium sequestration that
would have influenced muscle fatigability (Kampe &
Peters, 2013). In spite of this, there were no
significant differences evident in twitch force recovery
in the presence of DHT relative to Group 1, showing
that DHT played no significant role in returning the
force production capacity of fatigued gastrocnemius
back to base-line pre-fatigue levels.

observed that the recorded half-relaxation time ( RT)

offered no statistical difference in terms of the
contractility of muscles regardless of the presence or
absence of androgens, with evidently our recorded
means (fig. 5) indicating that the time to fatigue was
overall insignificant between groups. Although in
evident disagreement with our hypothesis, these
results were in agreement with the observations of
Fraysse et al. (2014). In spite of the fact that a
different animal model was used, they indicated that
the DHT-induced changes in the contractile properties
of the extensor digitorum longus (EDL) fibres of mice
remained unchanged between wild-type and AR
deficient male mice. This moreover indicated that DHT
played no definitive role in the influence of excitationcontraction (EC) coupling affecting the potential
upstream alterations in calcium release and
sequestration in both whole muscle and muscle fibre.
Nonetheless there are several studies in which
significantly larger isometric forces and RT were
evident in androgen-rich male bullfrogs relative to
that of the androgen-deficient female bullfrogs
(Peters & Aulner, 2000; Clark & Peters, 2006), when
comparing the amount of physiologically circulating
DHT between the two sets. One study furthermore
indicated that DHT interacts with an androgensensitive receptor via the MAPK pathway to regulate
and facilitate force production in mouse skeletal
muscle fibres (Hamdi & Mutungi, 2010).

In accordance with other studies (Kampe & Peters,

2013; Fraysse et al., 2014), acute effects of androgens
such as either testosterone or DHT had no effect in
influencing maximal force generation or fatigue
resistance. In either studies, the responses to the
administration of androgens showed a selectivity for
dimorphic muscles, and furthermore suggested that
variability existed in not only the myosin isoform
present in different muscles such as the tibialis
anterior (TA) or flexor carpi radialis (FCR), but also in
the pattern of up-regulation and down-regulation of
AR in breeding and non-breeding seasons
respectively. This was evident particularly in the
amphibian model (Melichna et al., 1972; Peters &
Aulner, 2000). Consistent with our results (Fig.2)
showing that DHT had no significant effect in forcecontractile properties, it must be noted that our
specimens were predominantly sampled during the
non-breeding seasons (Markula, Csurhes & HannanJones, 2010). Nonetheless, there were relatively
similar maximal twitch force amplitudes evident
across all four groups which suggested that there was
a limited interaction between the physiological DHT
concentrations administered (Harvey et al., 1997;
McCoy et al.,2008) and AR in all nerve-muscle
preparations based upon the literature. Additionally,
similar to our results (Fig.5), Kampe & Peters (2013)

Although we did not conduct accompanying

intracellular measurements of calcium in order to
assess the extent in which DHT and AR interacted in
the presence of flutamide, it is possible that DHT
facilitates its acute, non-genomic effects via a
different receptor independent to that of the AR
pathway. As our results suggested, there were no
significant changes evident in both the percentage of
force recovered following the recovery period, as well
as any significant DHT-mediated increases in maximal
twitch force evident. This therefore provided limited
scope to observe any potential abolishment of DHTmediated increases in force amplitude within the
presence or absence of flutamide.
Regardless, the lack of statistical significance in our
data between Group 2 and Group 4 does not dismiss
the independence of DHT from the AR pathway, with
previous literature also similarly suggesting an avenue
for further recommended research (Steinsapir et al.,
1991; Gorczynska & Handelsman, 1995; Bedrin et al.,
1997; Tvorogov & Carpenter, 2002; Bonaccorsi et al.,
2004; Sen et al., 2010). Nonetheless, Hamdi &
Mutungi (2011) suggested that DHT interacted with
both AR and Epidermal Growth Factor Receptor
(EGFR) simultaneously, thereby mediating its DHTinduced increases in muscle contractility via

downstream intracellular calcium releases from

Inositol Triphosphate (IP3) sensitive pools of SR (Oliver
et al., 2013). However, the downstream consequences
of this mechanism targeting EC coupling did not
translate into a significant increase in maximal twitch
force observed (fig.2). This suggests therefore that no
measurable difference in intracellular calcium
concentrations were significantly affected as our prefatigue and post-fatigue force amplitudes showed no
significant difference between each group (fig. 4).
Nevertheless, as our results suggested (fig. 6)
following the recovery period, the force-producing
capacity of the nerve-muscle preparations of each
group showed significant, ongoing reductions relative
to that of the baseline maximal amplitude. This is in
agreement with Jones (1996) and Edwards et al.
(1977)s description of postcontractile depression
(PCD) (Westerblad & Lnnergren, 1986) observed in
association with a markedly reduced and prolonged
depression in force production. This was evident in
cases where repeated short-tetani at low stimulation
frequencies were employed. In addition, similarly to
previous other studies that used the amphibian and
the rat model (Westerblad, Duty & Allen, 1993;
Callahan, She & Nosek, 2001 respectively), our results
indicated some limited but non-significant recovery
(Fig. 3 & Fig. 4) in muscle twitch force production. This
was furthermore indicative of the linkage between
PCD and the inhibition of the SR calcium release
properties that would have negatively influenced the
cross-bridging component of EC coupling mediated via
a reduction in cytosolic calcium concentrations
(Westerblad & Lnnergren, 1986; Lnnergren &
Westerblad, 1987; Howlett, Stary & Hogan, 2001).
As suggested by Liu et al. (2008), androgens would
therefore work to combat the effects of fatigue
observed via an inhibitive mechanism which would
potentially affect the activity of sarco-endoplasmic
reticulum calcium ATPases (SERCA), therefore having
a preventative action on calcium re-uptake which
would prolong the overall higher intracellular calcium
concentration. This would facilitate increased and
continual cross-bridging via troponin c interactions. As
this did not translate into a significant increase in the
time to fatigue between Group 1 and Group 3 in the
present study, it is evident that DHT had only a
minimal role in reversing the fatiguing-related
depression in intracellular calcium concentrations,
thereby refuting our hypothesis. It is recommended
that further studies examining the acute effects of
DHT using amphibian whole muscles should be
conducted within this context.
Limitations. It must be noted however that as we
recorded measurements following the fatiguing

protocol and the recovery period, the presentation of

an anoxic core may have influenced the fatigability
and synaptic efficiency (Reigner & Herrera, 1993) of
some nerve-muscle preparations in cases that
involved equipment failure. In these cases, reexamination of the nerve-muscle preparation was
required, thereby exposing the nerve-muscle
preparation to the protocol twice. This could
potentially result in the development of diffusion
gradients of oxygen and potassium affecting the
propagation of action potentials (Ruff, Simiocini &
Stuhmer, 1987; Jones, 1996) and subsequently
negatively skewering twitch force recovery peaks
(fig.3). This was observed in nerve-muscle preparation
13 (fig. 3). These findings were in support of the
observations of Barclay (2005), whereby the
development of an anoxic core in mouse soleus
occurred within 60 seconds at 20C following the
exposure of the muscle belly to repeated electrical
stimulations within a muscle bath similar to that used
in the present study.
Nonetheless, as indicated by previous literature
(Jones, 1996; Keeton & Binder-Macleod, 2006; Allen,
Lamb & Westerblad, 2008), use of short, intermittent
tetanic stimulations in lieu of our use of a constant
frequency train stimulation (CFS) would have also
provided a more effective basis for inducing
prolonged force depression in the nerve-muscle
preparations. Previous studies have observed that use
of CFS had a tendency to induce high-frequency
fatigue (HFF) whereby muscle preparations exhibited
rapid force production recovery subsequent to fatigue
(Jones, Bigland-Ritchie & Edwards, 1979; BiglandRitchie & Woods, 1984). This would be ineffective in
investigating the effects of DHT as HFF does not relate
to the changes in calcium transient and EC-coupling
associated with LFF. In addition, previous studies have
successfully examined the induction of PCD in
amphibian models via the use of a non-CSF
stimulation protocol with a set number of pulses
administered in staggered trains, such as 70Hz train of
pulses repeated at 0.3-0.8Hz with a 0.5s duration
conducted in one study (Westerblad & Lnnergremn,
1986). The administration of a 200 msec train of
stimuli at 30 pulses per second (pps) every two
seconds also managed to successfully induce PCD in
samples of b. marinus (Peters & Aulner, 2000; Kampe
& Peters, 2013). In retrospect, consideration of these
protocols could have greatly assisted in reducing some
of the variability evident in twitch force recovery for
Group 2 and Group 4 (fig.3 & fig.4)
A larger and more diverse sample set would have also
benefited the present study in terms of reducing
variability and achieving statistical significance. Use of
a combination of female, wild-type male and

orchidectomized male b. marinus samples would have

better illustrated the interaction between DHT, AR
and muscle contractility. Given that our sample set
consisted of exclusively males toads derived from
predominantly non-breeding season, analysis of the
effects of DHT in a sample population with either
reduced or non-significant amounts of pre-circulating
DHT would provide a better basis for understanding
the effects of DHT in affecting the RT and the
restoration of twitch force following LFF.
Conclusion
Our results indicated that DHT had no significant role
in delaying fatigue onset and that it had little effect on
the contractile properties of whole muscle
gastrocnemius in male b. marinus. In rejection of our
hypothesis, there were no significant increases to the
maximal force produced from the contracting nervemuscle preparation in the presence or absence of
DHT. In addition, no statistical significance providing
insight into the interaction between DHT and the
androgen-sensitive receptor pathway was evident,
and could therefore not support the mechanistic
action of DHT purported by existing literature. This
suggests that DHT would have a limited therapeutic
scope for the symptomatic relief and rehabilitation of
muscle-weakness in patients.

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