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mise à jour du
2 septembre 2002
Hormones And Behavior
1994; 28, 155-164
lexique
Behavioral effects of an antiandrogen in adult male Rhesus Macaques (Macaca mulatta)
BL Deputte, J Johnson, M Hempel, G Scheffler
Wisconsin Regional Primate Research Center, University of Wisconsin at Madison,
CNRS Université de Rennes, Station Biologique de Paimpont, 35380 Plélan le G
Index de tous les travaux du Prof BL Deputte

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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.
 
Threshold for behavioral response to testosterone in old castrated male rhesus macaques CH Phoenix and KC Chambers
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  
Testosterone Propionate Treatment of an XY Gonadal Dysgenetic Chacma Baboon.Bielert C
yawns-canines
credit photo : "Asif A. Ghazanfar and Aristides Arrenberg"
Max Planck Institute for Biological Cybernetics
Tuebingen; Germany.
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