In adult nonhuman primates many behaviors
are either more frequent in one gender than in
the other or even are specific to only one
gender. For some of these behaviors the role of
sex hormones has been demonstrated. Sex hormones
are assumed to have an organizing effect on
brain structures during the fetal life (see Goy
& McEwen, 1980). Sex dimorphic behaviors may
develop as early as the first year of life or
may appear only at puberty. Yawning in rhesus
and other primates is one of some behaviors
which become obviously dimorphic at puberty,
being much more frequent in adult males than in
any other age-sex classes (Hadidian,
1980; Deputte 1978,
1994).
The steroidal antiandrogens, such as
cyproterone acetate, have been mostly used in
the therapy of androgen-dependent disease, such
as prostate cancer (Neuman et al., 1977), and in
psychiatry (Horn, 1977). But the steroidal
antiandrogens have a twofold action, and both
antiandrogenic and estrogenic effects can be
obtained. Non-steroidal antiandrogens do not
present this estrogenic effect, so their use
could be more pertinent to study specifically
the failure of androgenic stimulation. These
antiandrogens, also called pure antiandrogens,
are for example flutamide, hydroxyflutamide,
RU23908, RU22930 (Raynaud et al., 1977). Their
effects have been investigated, in vivo, in sex
differentiation and for some behavioral effects
in rats (Neri et al., 1972; Gray, 1977,
respectively), for their antiprostatic
properties in dogs (Neri & Monahan, 1972)
and in baboons (Müntzing et al., 1974), and
in vitro for their binding properties in brain
tissues (Sholl, personal communication). As far
as we know, in primates, no behavioral studies
have been reported. The aim of this pilot study
is to investigate behavioral effects of a
nonsteroidal antiandrogen, hydroxyflutamide, and
to find out the doses necessary to block the
actions of androgens. We chose Hydroxyflutamide
rather than Flutamide as the hydroxy form is the
active metabolite in vivo (Tucker et al., 1988),
and Sholl (personal communication) has shown, in
vitro, that it is the most active form.
Yawning behavior has been chosen as a
behavioral indicator of androgen activity as its
frequency has been shown to decrease when adult
males are castrated and increase again after
substitutive injections of testosterone
(Bielert, 1975; Goy & Robinson, 1982). Both
the decline and the increase can be obtained
within a few days to a week following these
hormonal manipulations. Thus this behavioral
system has the clear advantage of responding
rapidly to hormonal changes.
SUBJECTS AND METHODS : Six adult male
rhesus macaques, Macaca mulatta, castrated
wiihin 3 months after birth were used. At the
start of the experiment, their weights ranged
from 6.3 te, 12.7 kg. The major experiment
consisted of two series of tests distributed
among four distinct phases. Both series began
with a Baseline phase during which the castrated
males were tested without exogenous androgen.
After completion of Baseline tests the Induction
phase commenced during which monkeys received
daily intramuscular (I.M.) injections of
testosterone propionate (TP), as indicated under
Results. The third phase differed for the first
and second series, but in both series the aim
was to reverse the effects of the Induction
phase. In the first series, reversal was
accomplished by discontinuing the TP injections.
In the second series, TP was continued, but
along with injection of androgen,
hydroxyflutamide (OHF, Shering Corp.,
Bloomfield, NJ) was injected daily. Reversal
phases lasted until the Baseline frequency of
yawning had been reestablished. Following
reestablishment of Baseline values, a
Re-induction of yawning behavior was initiated
as the fourth phase of the series. The four
phases are referred tc, as Baseline, Induction,
Reversal, and Re-induction in the order in which
they occurred in each series.
A second experiment was run in order to
determine whether the antiandrogen was effective
at a higher, supraphysiological dose of TP (0.25
mg/kg). This second experiment did not include
an independently determined Baseline phase, so
results are reported only for Induction,
Reversal and Re-induction for both the first
(withdrawal of TP) and second (administration of
OHF) series.
The hydroxyflutamide was suspended in an
aqueous vehicle composed of 0.5%
carboxymethylcellulose of low viscosity in 0.9%
saline solution. Solutions of 100 mg/ml of OHF
were used. Because we were using this
antiandrogen in behavior4l experiments,
subcutaneous injections were given rather than
intramuscular injections performed on
anesthesized subjects as described in
Müntzing's article on baboons
(Müntzing et al., 1974). Injections were
given around 4:30 pm, after the observation
sessions.
Behavioral Test Procedures : During a
standardized 15-min test, each male was paired
with the same adult female. Her hormonal
condition was kept constant by weekly
intramuscular injections of 0.5 mg of estradiol
cypionate (Legere Pharmaceuticals, Scottsdale,
AZ). Each pair was observed in a large pen M 6"
1/4 x 7' 3" 1/8 X 7' 4" 1/2.) Between each test,
the pen was carefully washed. The order in which
males were tested was varied for each session
(testing day). Tests were given 4 or 6 days each
week. The behavioral repertoire we used included
interactive and self-directed behaviors. The
interactive class (13 items) referred to
behavioral categories such as proximity,
affiliative behaviors, sex and aggression.
Self-directed behaviors (9 items), quoted for
the male only, referred to indicators of arousal
states of the male, such as scratching,
body-shaking, pacing, self-biting, displaying,
and yawning, but the quantitative analysis
presented here bears only on yawning behavior.
Within each series of experiment, the means over
the six males were computed for each session.
For a given phase, the mean was the mean over
the different sessions within this phase. The
Mann-Whitney U test was used for evaluating the
différences between means. The protocol
for the study was approved by the Animal Care
Committee.
RESULTS AND DISCUSSION The yawning
frequency of castrated males significantly
increased after injections of exogenous
testosterone. In the course of the study, during
induction or recovery phases, males had been
injected with three different doses of TP, high
= 0.50 mg/kg, medium = 0.25 mg/kg, and low =
0.10 mg/kg. Yawning frequency was not
differentially significantly influenced by the
three doses of TP (Kruskal & Wallis: H =
3.448, p > 0.10). This suggested that the
relationship between testosterone and yawning
was not a direct one. Indeed, no significant
correlations were found between yawning
frequencies and the residual circulating levels
of testosterone when blood drawings were
performed after two different baseline phases (p
= -.100 and 0.43, n.s.), or after periods of
induction with testosterone (rho ranged from
-.257 to +.500, n.s.). In order to determine the
dose of OUF that would be necessary to mimic
effects of withdrawal. of TP, several trials
were made with different combinations of
01-IF/TP ratios and varying durations of OUF
injections. On one trial, after an induction
phase with 0.25 mg/kg of TP, injections of 5
mg/kg of OHF twice weekly for 3 weeks were
evaluated. This dose constituted a ratio OUF/TP
of 20. In addition one series of injections of 5
mg/kg of OHF for 7 days, one series of
injections of 15 mg/kg of OHF for 3 days, and
one series of injections of 20 mg/kg of OHF for
3 days were conducted. All these combinations of
dose x duration were ineffective, and all except
the last one yielded lower overall doses than
those injected intramuscularly to baboons by
Müntzing et al. (1974), Le 5 mg/kg 3 times
weekly for 4 weeks. All these dose x duration
tests failed to induce a statistically
significant decrease in yawning frequency. In
addition an unexpected resurgence of sexual
behavior was observed during the 3day
antiandrogen phase when the males were injected
with a dose of 15 mg/Kg of OHF. Two males
mounted the female and one of them performed
intromissions.
Experiment 1: Effects of a Low Dose
of TP. Assay of blood samples collected 24 hr
after a single injection of 0.10 mg/kg of TP
revealed concentrations well within the normal
physiological range (Robinson et al., 1975).
Individual residual values in our subjects
varied from 1.28 to 6.27 ng/ml (mean = 3.8, c.v.
= 48.79).
First Series: Reversal by Withdrawal of
Testosterone The Baseline of the first series
started after males had not received any
androgens for 16 days. The Baseline lasted 7
days. Then the males received 6 injections of TP
(.10 mg/kg) during the Induction phase. The
frequency of yawning increased fivefold (Umw =
1, p = 0.014; Fig. 2). The Reversal phase lasted
10 days. Immediately upon the cessation of TP
injections the yawning frequency decreased
straightforward. The mean frequency of yawning
during the first 4 days of Reversal did not
differ significantly from the yawning frequency
during the Induction phase Umw = 5, p = 0.243;
Fig. 2), but during the last 4 sessions of the
Reversal phase, the yawning frequency remained
low and differed significantly from the level
during the Induction phase but not from the
Baseline level (Uw = 1, p = 0.029 and Umw = 3,
n.s.; respectively, Fig. 2). The frequency of
yawning started to increase sharply as soon as
after the first TP injection of the Re-Induction
phase (Fig. 2) and differed significantly from
the frequency of yawning during the last days of
the Reversal phase but not from the yawning
frequency during the Induction phase (Umw = 0, p
0.014 and Umw = 5.5, p > 0.243,
respectively).
Second Series: Reversal by Injection of OHF
: A new baseline was established after 10 days
without any injections. The Induction phase
lasted 6 days during which the same daily dose
of 0. 10 mg/kg as that of the first series was
injected. The yawning frequency rapidly showed a
significant increase (Umw = 0, p = 0.004; Fig.
2). During the Reversal phase a dose of 8 mg/kg
of OHF was injected subcutaneously,
simultaneously with the 0.10 mg/kg of TP. The
yawning frequency started to decrease as soon as
the first injection of OHF. However during the
first 5 days of the Reversal phase the yawning
frequency did not differ significantly frorn
that of the Induction phase (Umw = 8.5, p >
0.210; Fig. 2). But as the yawning frequency
continued to decrease, during the last 6 days,
it reached a level not significantly different
than those of the Baseline phase and
significantly lower than that of the Induction
phase (Umw = 7.5, p > 0.155 and Umw = 4, p =
0.048 respectively; Fig. 2). The yawning
frequency reached during the last days of the
Reversal phase remained stable during the first
5 days of the Reinduction phase when only TP was
injected and did not differ from the Baseline
level (Umw = 9.5, p > 0.452, and Umw = 4, p =
0.095, respectively; Fig. 2). The yawning
frequency did not start to increase steadily
during the Re-induction until the seventh
injection. On the seventh and subsequent tests
the yawning frequency equaled that of the
Induction phase and differed from the level of
the first five days of the Re-induction phase
(Umw = 7, p = 0.278 and Umw = 1.5, p = 0.029,
respectively;
Experiment 2: Effect of High Dose of
Testosterone Propionate Twenty-four hours after
the ninth injection of .25 mg/kg TP, residual
levels of circulating testosterone ranging from
1.3 to more than 3 times the mean level induced
by the lower dose were found. The average
concentration inducect by this suprapysiological
dose was 16.3 ng/ml compared with an average of
3.8 ng/ml when only .10 mg/kg was injected.
First Series: Reversal by Withdrawal of TP
(0) Inasmuch as several doses of OHF as well as
several différent durations of injections
of antiandrogen had been tested just prior to
this experiment, no new baseline level was
determined. Thus, at the start of the first
series of the second experiment, the frequency
of yawning was over seven yawns per individual
(Table 1) and continued injections of TP did not
significantly increase yawning above this level.
As soon as the first day without exogenous
testosterone, the yawning frequency dropped. It
decreased significantly during the first 4 days
of the Reversal phase (Umw = 0, p = 0.028; Table
1) and reached a low level by the 4th day of
Reversal (3rd test; Table 1). The Reversal phase
lasted 10 days. As soon as the injections of
testosterone were resumed, during Re-induction,
there was a significant increase of yawning over
that seen during the final test of Reversal.
Moreover the Re-induction phase did not
significantly differ from that of the Induction
phase (Umw = 2, p = 0.114; Table 1).
Second Series: Reversal by Injection of OHF
: During the 6-day Induction phase the average
yawning frequency was 5 yawns per individual.
The yawning frequency of the first 4 days of the
Reversal phase with OHF (20 mg/kg) was
significantly lower than that of the Induction
phase (Umw = 0, p = 0.014; Table 1). During the
first 5 days of the Re-induction phase the
continued injections of 0.25 mg/kg of TP failed
to increase the yawning frequency which remained
similar to that of the last four days of the
Reversal phase (Umw = 2.5, p > 0.25; Table
1). After 1 week of daily injection of TP, the
yawning frequency started to increase again. So
the yawning frequency of the last 4 days of the
10-day Re-induction phase was significantly
higher than that of the first 3 days of this
phase (Umw = 0, p=0.028; Table 1).
Our experiments show clearly that a pure
antiandrogen is able to inhibit the positive
influence of the androgens on occurrence of
behaviors such as yawning when the ratio of
OHF/TP equals 80. After injections of exogenous
testosterone in castrated males, OHF injected
simultaneously with TP provokes a decrease in
yawning frequency similar to that of a
withdrawal of TP. The effective overall doses
used in this study, i.e the dose x duration
combination, exceeded by a factor over 1.5 (1.7
and 2.3) those shown effective in decreasing the
weight of the caudal prostate in baboons
(Müntzing et al., 1974). This difference
could be likely attributed to the route of
injection of OHF, subcutaneously in this study,
intramuscularly in Müntzing's study.
This difference in dosage requirements could
also be attributed to differential effects of an
antiandrogen on anatomical structures or on
behavior. It should be remembered that the
yawning/androgens relationship is only
probabilistic and that yawning could be
influenced also by adrenal activity (Deputte
1994). That fact that low and high doses of
testosterone could induce similar increases in
yawning suggests that the most important factor
could be the way the T.P. is metabolized rather
than the dose injected.
Compared to the effect of a withdrawal of
TP, injections of OHF had a slower but a longer
lasting effect. This second feature is
particularly noticeable. An inhibiting effect
which lasts at least 5 days after the last
injection of OHF can be observed in our data.
The delay in the reappearance of the behavior is
in contrast to the rapidity with which it is
restored when TP injections are resumed
following a withdraw. This could be due to the
nature of the OHF injections, being associated
with both the type of the vehicle and with a
"site effect.- In addition, the fact that the
drug was injected as a crystalline suspension
rather than completely dissolved in its vehicle
probably resulted in a slow diffusion from the
injection site into the circulation.
There is a large interindividual variability
in the efficacy with which OHF decreases the
frequency of yawning. However all males but one
reached the basal level of yawning for at least
two tests during both experiments, either during
the antiandrogen treatment or during the first
days of the Re-induction phase following a
reversal by means of OHF. This indicates that
the différences in effectiveness cannot
be accounted for by individual differences in
basal frequencies of the behavior.
This study shows that a dose of OHF equal to
8 mg/kg is able to inhibit some of the
behavioral effects induced by levels of
circulating testosterone ranging from 1.3 to 6.8
ng/ml. These effects of a pure antiandrogen
could be obtained from subcutaneous injections
using a low-viscosity vehicle. These results are
encouraging to the possibility of designing
other behavioral studies using pure
antiandrogene.
Biology of Reproduction, Vol 35, 918-926,
1986 by Society for the Study of
Reproduction
The sexual behaviors of old, intact (N = 5)
and old, castrated (N = 6) rhesus macaque males
were compared in six series of pair tests with
receptive females. The castrated monkeys were
tested when untreated and when given five doses
of testosterone propionate (TP; 0.004, 0.016,
0.064, 0.256, and 1.024 mg/kg of body weight) in
consecutive months. The serum testosterone (T)
level was determined for each male before and
after each series of tests. When untreated, none
of the castrated males ejaculated, and
yawning was significantly less in these
monkeys than in intact males-no other
behavioral measures differed significantly.
Within 2 weeks of daily injections of 0.004 mg
of TP/kg, two males ejaculated, and all
differences in measures of ejaculation were
eliminated. A third male ejaculated after 1 week
of treatment with 0.016 mg of TP/kg. Yawning
values did not differ during and after
treatment with 0.064 mg of TP/kg. Although final
mean serum T levels were six times higher in
castrated (24.3 ng/ml) than in intact males (4.2
ng/ml), sexual performance levels did not exceed
those of intact males.
« It is
ironic that testosterone "the male sex hormone,"
is more closely associated with the yawning rate
than with the mounting or intromitting rates
» Charles Phoenix
Sexual
steroids
exert several effects on both central
dopaminergic and oxytocinergic systems by acting
either at the genomic or membrane level
