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Effects of testosterone on the behavior of male cynomolgus monkeys (Macaca fascicularis)
HORMONES
AND
BEHAVIOR
19, 265-277 (1985)
Effects of Testosterone on the Behavior of Male Cynomolgus Monkeys (Macaca fascicularis) DORIS
ZUMPEAND RICHARD P. MICHAEL’
Department of Psychiatry, Emory University School of Medicine, Atlanta, Georgia 30322 and The Georgia Mental Health Institute, Atlanta, Georgia 30306 To determine the threshold doses of testosterone propionate (TP) that cause clear-cut behavioral changes in the sexual behavior of castrated male cynomolgus monkeys, observations were made on three males during successive S-week treatment periods while they received daily subcutaneous doses of 100 pg TP increasing in octaves to 25.6 mg TP. Males were tested with each of the same two ovariectomized, estrogen-treated females (6 pairs, 330 I-hr behavior tests). To mimic the diurnal plasma testosterone rhythm, TP injections were given at 1600hr and blood samples were obtained at 0800hr (141 samples). Male ejaculatory activity increased at the threshold dose of 200 pg TP per day giving plasma testosterone levels of 830 ng/lOO ml, which is in the physiological range of 6001600 ng/lOO ml for intact males. This threshold dose was eight times higher than in rhesus monkeys on a dose per kilogram body weight basis. There was a further marked increase in ejaculatory performance at higher doses (6.4 to 25.6 mg) giving supraphysiologicaf plasma levels of 4000-9000 ng/lOO ml. There were individual differences in the behavioral changes occurring with TP treatment, and the female partner modulated the effects. These findings were generally similar to those obtained with male rhesus monkeys, but certain species differences were noted. Q 1985 Academic Press, Inc.
Much of what is currently known about the effects of androgens on the sexual behavior of male primates has been derived from castration and hormone replacement studies with male rhesus monkeys (A4ucaca muluttu). Castration decreasesboth the ability to intromit and the frequency of ejaculations; although individual males may continue ejaculating for more than 2 years after surgery. Replacement treatments with high doses of testosterone propionate (2-10 mg/day) restore male sexual activity in a 6- to IO-week period (Michael, 1972; Michael, Wilson, and Plant, 1973; Phoenix, Slob, and Goy, 1973; Phoenix, 1974; Michael and Wilson, 1974). More recently, we reported that very small doses of testosterone propionate (50-100 &day), which gave plasma testosterone levels in the 200-450 ’ To whom correspondence should be addressed: Department of Psychiatry, Emory University School of Medicine, Atlanta, Ga. 30322. 265 0018-506X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
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AND MICHAEL
r&100 ml range, also increased the sexual activity of male castrates (Michael, Bonsall, and Zumpe, 1984). These plasma testosterone levels were below the physiological range for intact males in our laboratory. Individual differences between males and between females, as well as the hormonal status of the latter, exerted powerful modulating effects on the behavioral responses to testosterone treatment. To our knowledge, no systematic castration and hormone replacement studies have been conducted with any male anthropoid primate other than the rhesus monkey, and the present study was undertaken to begin to fill this gap using the closely related cynomolgus monkey (M. fuscicularis). The cynomolgus monkey has interest for several reasons. It is being increasingly used to replace the rhesus monkey in studies on the hormonal control of behavior now that young feral-reared rhesus monkeys are so difficult to obtain. It is taxonomically close to the rhesus but shows some significant differences; it does not breed seasonally (Dang, 1977; Kavanagh and Laursen, 1984), it is less sexually dimorphic, and its copulatory behavior consists primarily of single mount ejaculations (Zumpe and Michael, 1983). To facilitate comparisons with the rhesus monkey, in the present study we administered increasing amounts of testosterone propionate subcutaneously to castrated male cynomolgus monkeys, beginning at doses of 100 pg/day and increasing in octaves to doses of 25.6 mg/day. Behavioral interactions with estrogen-treated females were conducted on a regular basis and plasma testosterone levels were measured by radioimmunoassay to permit correlations with sexual behavior. METHODS Animals. Four male (weighing 5.0-5.2 kg) and two female (weighing 3.1-4.7 kg) cynomolgus monkeys were obtained as adults through dealers directly from Malaysia. Males were castrated 3 months before the start of this experiment, and females had been ovariectomized over 1 year earlier. Unfortunately, one male died during the early months of this study and these results were discarded. Operative techniques were identical to those described in detail elsewhere for rhesus monkeys (Michael and Wilson, 1974). Each male received a pair of Silastic testicular prostheses after castration to restore the appearance and stimulus properties of the scrotal sac. Animals were maintained throughout in individual cages together in a room with windows so that the artificial lighting was supplemented with natural daylight. Temperature was maintained between 20 and 24°C. Food consisted of Purina monkey chow with vitamin supplements and water was available ad libitum. Hormone treatments. Following a lO-week period of testing (two periods each of 5 weeks) to establish behavioral baselines, all males received successive 5-week periods of treatment with testosterone propionate (TP) subcutaneously in 0.2 ml oil at the following dose levels: 100, 200, 400,
TESTOSTERONE
AND
BEHAVIOR
267
and 800 pg, 1.6, 3.2, 6.4, 12.8, and 25.6 mg per day. All injections were given at 1600 hr, and the first injection of each new treatment was given on the afternoon (Friday) after the last behavioral test of the preceding treatment, so that three injections were given before the first behavioral test of the new treatment (Monday). Beginning 3 months before the start of the study, females received daily subcutaneous injections of 5 pg estradiol benzoate in 0.2 ml oil at 0800 hr. Plasma samples and hormone assays. Starting 3 weeks before the first hormone injection, 3 ml blood was obtained once a week at 0800 hr from the saphenous veins of the untranquilized males that had been previously adapted to the procedure (141 samples). Results were compared with plasma testosterone levels in three 0800 hr blood samples obtained from each male several weeks before castration (9 samples). All samples from a given male were initially analyzed simultaneously. However, owing to the unusually high hormone levels reached in this study, samples from the three highest treatment regimes were reanalyzed at higher dilutions to obtain more accurate estimates. Plasma testosterone levels were determined by radioimmunoassay on 40 ~1 plasma (Bonsall, Baumgardner, and Michael, 1976) using a high-affinity antiserum to testosterone-3-(C)carboxymethyl)oxime-bovine serum albumin produced in these laboratories. The only major cross reaction was with dihydrotestosterone (40%), and this elevated testosterone estimates in plasma samples by up to 16%. The coefficients of variation between duplicates were 7.4% (within assays) and 12.7% (between assays). Water blanks read 3.5 pg and the blank reading was not subtracted. Behavioral testing. Observations were made on oppositely sexed pairs of animals in quiet, isolated rooms from behind one-way vision mirrors in special observation cages (1.19 m wide by 1.07 m deep by 1.14 m high) into which first the male and then the female was introduced at the start of each test session. Tests were of 60 min duration and were conducted 3 days a week (Monday, Wednesday, and Friday). On each test day, two males were each tested with one of the two females, and pairings alternated so that each of the six pairs was tested once a week. No animal was tested more than once per test day. Both tests on a given day were conducted at the same time between 0900 and 1200 hr. The findings reported here are therefore based on a total of 330 behavior tests with six pairs of animals involving three males and two females. Definitions and terminology. The basic descriptions of the behavior observed during tests have been given elsewhere (Zumpe and Michael, 1983). Twenty-nine of the behavioral measures routinely scored were analyzed statistically, but results for only 12 of these are given here: (a) number of ejaculations per test (potency)-the characteristic behavioral response, typical of ejaculation in intact males, performed in coitu while mounting the female; (b) time to first ejaculation-time in seconds from
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the start of the test to the first ejaculation (a default value of 3600 was given where ejaculation did not occur); (c) number of mounts per test; (d) number of male mounting attempts per test-sum of male initiated mounts and attempts to mount that were refused by the female; (e) first mounting attempt latency-time in seconds from the start of the test to the first male mounting attempt (a default value of 3600 was given where there were no mounting attempts); (f) number of intromitted thrusts per test; (g) number of unintromitted thrusts per test; (h) total thrusts per test-sum of intromitted and unintromitted thrusts per test; (i) percentage intromitted thrusts-intromitted thrusts expressed as a percentage of total thrusts per test; (j) male grooming time-time in seconds spent by the male in grooming the female; (k) female grooming time-time in seconds spent by the female in grooming the male; (1) number of male yawns per test (Goy and Resko, 1972). Statistical treatment of results. The significance of differences between plasma testosterone levels during pretreatment and during the different testosterone treatments was assessedby a three-way analysis of variance for repeated measures using a treatment by sample by male design. For statistical purposes only, a male’s treatment mean was used in place of a missing sample value to fill a cell (9 of the 150 samples were not available). Differences between behavioral frequencies were analyzed using a treatment by female by male design, and for this purpose the lo-week pretreatment period was divided into two 5-week periods (Bl and B2). Individual treatment means were compared using the Scheffe test. The significance of associations between changes in plasma testosterone and in behavioral measures was tested by Spearman rank correlation coefficient. RESULTS Testosterone and Male Sexual Activity-Group
Results
The effects of increasing doses of TP on the plasma testosterone and sexual behavior of castrated male cynomolgus monkeys are shown in Table 1. There were highly significant changes in mean plasma testosterone levels at 0800 hr (16 hr after injection), which ranged between 414 and 9129 ng/lOO ml over the 100 pg to 25.6 mg dose range. Physiological levels in these three males when intact were between 600 and 1600 ng/ 100 ml at 0800 hr. Similar levels to these were produced in castrates by doses of 200 pg and 400 ,ug TP per day (Fig. 1). As plasma hormone levels increased, there were highly significant increases in ejaculatory performance. At the 200 pg TP dose, when plasma testosterone reached physiological levels, numbers of ejaculations increased abruptly while ejaculation times decreased equally sharply (Table 1). At the 3.2 mg TP dose level, there appeared to be a further increase in the number of
thrusts
grooming
time
1426 r 95
1085 + 160
3 f 1.4 0.2 2 0.08
3 2 1.4
1388 t
70
1197 + 134
6 f; 2.2 0.2 5 0.08
9 % 4.2
117
IOOS *
120
71 2 5.4 1.6 r 0.49
12.5
and 30 behavior
tests.
2044 c 92
820 + 121
92 k 3.0 5.0 + 0.98
100 + 13.6
6 + 1.9
34 zt 12.4 85 *
248 ? 11% 106 -c 13.5
II L 4.1 I20 zc 16.4
8.0 + 1.17
756 k 244 9.1 f 1.25
953 t 246 8.8 k 1.26 7.8 2 1.00
2464 k 144 1.7 f 0.20
1437 2 98 1.6 + 0.19
8~ ~g TP
Activity
O.%
9.4
122 2206 + 106
807 f
82 5 5.3 5.2 + 1.09
75 f
12 2 4.7
399 _c 201 87 + 11.2
6.0 f
1275 f 216 6.7 + 0.91
3385 ? 263 1.5 z 0.20
1.6 mg TP
TABLE 1 Levels and Sexual
400 fa TP
Testosterone
2074 ? 91
samples
1%3 + 136
1216 -c 174
66 k 4.8 0.7 + 0.29
69 2 8.6
37 e 6.9
18 * 9.7 107 2 9.8
5.2 2 0.56
81s 2 215 6.6 _f 0.67
830 * 113 1.5 + 0.13
200 fig TP
on the Plasma
of P-15 plasma
1700 z 84
1376 *
17 + 4.8 0.2 ? 0.09
20 + 5.4
76 2 10.9
84 k 13.2
0.60
701 2 256 % 2 12.4
3.8 f
857 + 281 94 t 14.8
4.1 2 0.66
2310 c 295 4.7 + 0.55
2637 + 254 5.0 + 0.75
100 pg TP
414 2 50 0.6 + 0.14
2
54 % 4 0.4 -t 0.11
Baseline
Propionate
* Fq.18 for plasma testosterone and F10.m for behavior. ’ Values in the bcdy of the Table are the means * SEM
(sed
bea Female
per test Male yawns per test Male grooming time
test % Intromitted
per
114 + 15.3
thrusts
per test Intromitted
6.4 + 0.92
380 k 183 117 + 15.3
attempt
per
0.09
1
Doses of Testosterone
3170 -c 192 7.3 _f 0.86
0.2 t
Baseline
of Increasing
latency (set) Total thrusts per test Unintromitted thrusts
test Fit mounting
(sea Mount.9 per test Mountiog attempts
cwmo ml) Ejaculations per test Time of ejaculation
Plasma testosterone
Effects
0.72
2264 I?: 133
2504 f
118
635 + 114
764 + 126
85 -c 8.1
2.7
89 + 2.9 5.5 k 1.19
10.1
10 f
246 * 166 95 2 9.2
6.3 2 0.91
68 * 5.9 6.8 -’ 1.22
72 z
28 -+ 6.4
248 t 166 101 5 12.0
5.0 t
139 2 87 7.2 2 0.74
548 -t 193 6.8 -t 0.92
6.4 mg TP
Cynomolgus
4149 k 278 2.6 t 0.14
Male
3712 + 243 2.2 2 0.19
3.2 mg TP
of Castrated
166 9.5
105 2458 z. 115
633 k
a7 2 3.3 4.0 + 0.87
90 e 8.2
1s 2 4.7
248 t 105 f
5.2 k 0.66
51 + 20 7.5 A 0.59
6 Pairs)”
5.2
8.1
844 ‘c 116 2191 + 110
45.4 3.4
6.0 89 ” 3.8 5.8 -+ 1.23
9.3
7.1
7 Et 1.8 86 t
1.3 0.5
126 5 120 93 2 9.6
2.3
11.7 1.9
134 + 120 6.7 -t 0.68 4.9 k 0.59
44.6 19.0
F*
9129 2 743 2.6 + 0.19
25.6mgTP
(3 Males.
5032 k 253 3.2 ? 0.14
12.8mgTP
Monkeys
co.001 <0.025
NS NS
NS
~0.001
P
270
! ! -I’1---_ ..-
ZUMPE AND MICHAEL
61 62
400~ b6m9 64mq 25.61rq 200~0 600~ 32mg I2.6mg
TESTOSTERONE PROPIONATE DOSAGE FIG. 1. Changes in ejaculations and plasma testosterone levels in three castrated male cynomolgus monkeys (6 pairs) given increasing daily doses of testosterone propionate (TP) subcutaneously. Data from 330 mating tests and 141plasma samples. In this and subsequent figures Bl and B2 = two successive S-week periods of pretreatment baseline. Vertical bars given standard errors of means. Interrupted horizontal lines indicate the upper and lower limits of the physiological plasma testosterone range for these males when intact.
ejaculations, and this was associated with plasma levels greatly in excess of the physiological range. Mounts, mounting attempts and mounting attempt latencies did not change significantly and no consistent patterns were observed, perhaps because these behaviors had not yet been affected by castration. However, intromitted thrusts increased significantly and unintromitted thrusts decreased significantly at 200 pg TP but did not change further at higher doses. Percentage intromitted thrusts increased significantly during the 200 pg TP treatment and reached a maximum with the 800 pg TP treatment. The numbers of male yawns, an androgendependent behavior in rhesus monkeys (Goy and Resko, 1972) increased markedly at the 800 pg TP dose. This increase reached statistical significance at the 1.6 mg TP dose but showed no further changes at higher dose levels. There were no significant effects of testosterone on measures of agonistic and social behavior, except for decreases and increases in male and female grooming times, respectively (Table 1). There was a positive association between changes in plasma testosterone and changes in ejaculations (rS = 0.93, N = 10, P < 0.001) and a negative association between plasma testosterone and times of ejaculation (r, = -0.90, N = 10, P < 0.001). Changes in plasma testosterone were also significantly associated with changes in intromitted thrusts, unintromitted thrusts, percentage intromitted thrusts, yawns, and male and female grooming times (r, = kO.70 to kO.85, N = 10, P < 0.0.5-0.01). There
TESTOSTERONE
AND BEHAVIOR
271
were no significant associations between changes in plasma testosterone and changes in total thrusts, mounts, mounting attempts or mounting attempt latencies (Y, = +0.21 to kO.47, N = 10, NS). Thus, ejaculatory performance was more closely related than any other behavioral measure to plasma hormone levels, including those reflecting the ability to obtain intromission. Testosterone and Male Sexual Activity-Individual Males Although plasma testosterone levels in the different males were generally rather similar at each dose level (F2,36= 0.57, NS), there were highly significant differences between males for all behavioral measures (F2,r0 = 5.8-209.0, P < 0.025-0.001) except percentage intromitted thrusts and male grooming times. Male 2 made far fewer mounting attempts and yawns (Fig. 2) and fewer intromitted and unintromitted thrusts (Fig. 3) in response to testosterone than either of the other two males. There were also individual differences in the behavioral response patterns with increasing levels of plasma testosterone. Males 1 and 2 showed a fairly sharp increase in numbers of ejaculations at the 200 ,ug TP dose. Ejaculations increased again with higher doses to reach maximal levels during the 12.8 mg TP treatment. Male 3 also showed a definite increase in ejaculations at 200 pg TP, but in this male ejaculations appeared to increase progressively over the entire dose range (Fig. 2). Individual differences were also apparent in numbers of intromitted thrusts and percentage intromitted thrusts (Fig. 3): male 1 showed a graded increase with dose which reached a plateau at the 800 pg TP level, whereas males 2 and 3 showed a sharp response at a threshold dose of 200 pg TP. It was of interest that male 3 showed a very abrupt increase in percentage intromitted thrusts at 200 pg TP but a graded increase in ejaculations over the whole dose range. Testosterone and Male Sexual Activity-Individual Females Both female partners were ovariectomized and treated with 5 pg estradiol benzoate per day. While there were no significant behavioral differences between them (F,,Z = 0.2-13.6, NS), there was nevertheless evidence that they modulated the behavioral effects of treating males with testosterone. Figure 4 shows the mean numbers of ejaculations and percentage intromitted thrusts received by each female from all males. With female A, there appeared to be a close relationship between changes in the two behavioral variables; percentage intromitted thrusts and ejaculations increased markedly at the 200 pg TP dose and tended to covary at higher doses (Fig. 4, left). With Female B, on the other hand, there was no increase in ejaculations between the 200 pg and 1.6 mg. TP doses although the percentage of intromitted thrusts increased markedly as they did with female A; this produced a clear dissociation between the two behavioral
272
ZUMPE AND MICHAEL
TESTOSTERONE
PROPIONATE
DOSAGE
FIG. 2. Comparison of changes in the sexual behavior and plasma testosterone levels of three castrated male cynomolgus monkeys given increasing daily doses of TP. There were broadly similar changes in ejaculations but clear individual differences in the levels of other behavioral indices. In this and Fig. 3, the data for each male were from 110 mating tests and 41-43 plasma samples. Symbols as in Fig. 1.
variables (Fig. 4, right). Some property of female B must have been responsible for this. DISCUSSION These findings were quite clear-cut and reached acceptable levels of statistical significance using analyses of variance, so that it is permissible to make some comparisons with data from similarly treated male rhesus monkeys (Michael et al., 1984). Ejaculatory activity increased in castrated male cynomolgus monkeys with 200 E.L~TP per day which gave plasma levels of testosterone in the physiological range for intact males. In rhesus
273
TESTOSTERONE AND BEHAVIOR MALE
I
MALE
2
MALE
3
c
IO0
50
0
TESTOSTERONE
PROPIONATE
DOSAGE
FIG. 3. Comparison of changes in three measures of thrusting activity and in plasma testosterone levels of three castrated male cynomolgus monkeys given increasingly daily doses of TP. Although there were marked individual differences, in all three males intromitted thrusts reached maximum levels at 800 pg TP, while unintromitted thrusts declined to very low levels at this dosage. The ability to obtain intromission (middle panel) was fully restored with the 800 pg TP treatment. Symbols as in Fig. 1.
monkeys, ejaculatory activity increased with 50 pg TP per day which gave plasma levels of testosterone well below the physiological range for this species. It should be remembered, of course, that male rhesus monkeys are about twice the body weight of male cynomolgus monkeys, and so the rhesus monkey may be regarded as considerably (eight times) more sensitive to testosterone with respect to ejaculation than the cynomolgus monkey. This difference may relate to the fact that the rhesus monkey, unlike the cynomolgus monkey (Mahone and Dukelow, 1979), is a seasonal breeder which undergoes testicular regression during the
274
ZUMPE AND MICHAEL FEMALE
a $ oI
A
FEMALE
B
T/i
;/‘,/‘l o--Q ) , ) , , 81 IoOpg 4C9q 82
, , , , , ,
L
Wnq 6.4mg ZbBmq 2ooJq BooJIg 32mq 12~6rn6
62
TESTOSTERONE
PROPIONATE
200~9 BOOM 32ma 12.6~ DOSAGE
FIG. 4. The identity of the female partner helped to determine the responses of castrated male cynomolgus monkeys to TP administration. With female A, ejaculations and intromitted thrusts were closely associated throughout the dose range. With female B, a dissociation occurred, and the ability to intromit was fully restored at 800 pg TP whereas ejaculations did not increase until 3.2 mg TP. Each female was tested with the same three males. Symbols as in Fig. 1.
nonmating season and is more behaviorally sensitive to very small increases in plasma testosterone when levels are low. However, higher doses (3.212.8 mg) giving plasma testosterone levels greatly in excess of the physiological ranges resulted in further increases in the ejaculatory performance of both cynomolgus and rhesus monkeys. The two species differed in their potency and in their ability to obtain intromission. Cynomolgus monkeys showed a sharp increase in percentage intromitted thrusts (10% increased to 90%) with 800 pg TP per day, while in rhesus monkeys this change was much smaller (0% increased to 25%) with even higher doses. We are not sure if this difference, like the higher potency of the cynomolgus monkey (generally two ejaculations per test instead of one), is a genuine species difference related to its single-mount ejaculatory pattern or due to the longer period in captivity of our rhesus monkeys. Since the intromissions of cynomolgus monkeys were fully restored with 800 pg TP, the increase in ejaculations at much higher doses of testosterone (12.8 mg) could not readily be ascribed to a peripheral action of androgen on the penis. The possibility that higher doses caused an increase in male sexual motivation received some support from the observation that, as dose levels increased from 400 pg to 25.6 mg TP, there was a significant decrease in mean numbers of intromitted thrusts to ejaculation (33.3 r 8.42 to 14.0 + 0.22) (F,o,zo = 5.9, p < 0.001). In this study, the dissociation between changes in intromitted thrusts and
TESTOSTERONE
AND
BEHAVIOR
275
ejaculatory performance was most obvious in tests involving female B (Fig. 4); suggesting that some property of this female was responsible for the effect rather than a hormone-dependent mechanism in the males. It is possible that female A was made fully receptive by 5 Fugestradiol benzoate per day while female B was not, because we have noted a similar dissociation occurring in male rhesus monkeys when they were paired with ovariectomized, untreated females. Hormone injections were given at 1600hr, 16 hr before blood sampling, to mimic as closely as possible the diurnal plasma testosterone rhythm of intact animals (Steiner, Peterson, Yu, Conner, Gilbert, TerPenning, and Bremner, 1980; Michael et al., unpublished observations). The diurnal rhythm of cynomolgus monkeys, like that of rhesus and bonnet monkeys (Michael, Setchell, and Plant, 1973; Plant, 1981; Kholkute, Joseph, Joshi, and Munshi, 1981; Mukku, Murty, Srinath, Ramasharma, Kotagi, and Moudgal, 1981), results in low daytime and high nighttime plasma testosterone levels. The 24-hr plasma testosterone profile following a single injection of testosterone propionate at 1600 hr was similar to that in rhesus monkeys (Michael et al., 1973). In two of the males, injecting 400 pg testosterone propionate at 1600 hr resulted in mean plasma testosterone levels of 1263, 864, 675, 650, and 607 ng/lOO ml at 2, 6, 16, 20, and 24 hr after injection. Testosterone levels in 0800 hr samples in our animals when they were intact ranged between 600 and 1600 r&100 ml, which is consistent with a morning mean of 9.1 rig/ml in another study (Steiner et al., 1980). Over the 1.6-12.8 mg TP dose range, plasma testosterone levels increased only modestly from about 3400 ng/lOO ml to about 5000 ng/lOO ml, perhaps because of changes in metabolism and clearance; but levels doubled to about 9000 ng/lOO ml with the 25.6 mg TP treatment. Adjusting for body weight differences, the cynomolgus and rhesus monkeys showed quite similar plasma hormone levels over the 100 pg to 12.8 mg dose range. These findings extend our knowledge of the effects of testosterone on behavior to another, closely related species of anthropoid primate. On a dose per kilogram body weight basis, the threshold dose of TP for ejaculation was eight times greater in the cynomolgus monkey than in the rhesus monkey. It should be noted, however, that “threshold dose” is not an absolute constant but may vary with time since castration, with treatment duration, with increasing or with decreasing doses, with return to baselines between doses, and with other differences in experimental design. In both species, ejaculatory activity increased progressively with increasing plasma testosterone levels, but effects were modulated by the identity of the female partner. Clear behavioral changes were observed over the physiological dose range, and in both species additional wellmarked increases in ejaculatory activity occurred when plasma testosterone reached supraphysiological levels. However, cynomolgus monkeys, unlike
276
ZUMPE AND MICHAEL
rhesus monkeys, showed no increase in ejaculatory performance when plasma hormone levels were below the physiological range, and their ability to intromit was fully restored at doses of TP well below those producing maximum ejaculatory performance. The male cynomolgus monkey offers considerable promise for studies on the hormonal control of behavior. ACKNOWLEDGMENTS This work was supported by USPHS Grant MH 19506, and general research support was provided by the Georgia Department of Human Resources. Both are gratefully acknowledged.
REFERENCES Bonsall, R. W., Baumgardner, D. G., and Michael, R. P. (1976). A computerized semiautomated radioimmunoassay for plasma testosterone. J. Steroid Eiochem, 7, KU858. Dang, D. C. (1977). Absence of seasonal variation in the day length of the menstrual cycle and fertility of the crab-eating macaque (Macaca fuscicularis) raised under natural daylight ratio. Ann. Biol. Anim. Biochim. Biophys. 17, 1-7. Goy, R. W., and Resko, J. A. (1972). Gonadal hormones and behavior of normal and pseudohermaphroditic nonhuman female primates. Rec. Prog. Horm. Res. 28, 707733. Kavanagh, M., and Laursen, E. (1984). Breeding seasonality among long-tailed macaques, Macaca fascicularis, in Peninsular Malaysia. Int. J. Primatol. 5, 17-29. Kholkute, S. D., Joseph, R., Joshi, U. M., and Munshi, S. R. (1981). Diurnal variations of serum testosterone levels in the male bonnet monkey (Macaca radiara). Primates 22, 427-430. Mahone, J. P., and Dukelow, R. W. (1979). Seasonal variation of reproductive parameters in the laboratory-housed male cynomolgus macaque (Macaca fuscicularis). J. Med. Primatol. 8, 179-183. Michael, R. P. (1972). Determinants of primate reproductive behaviour. In E. Diczfalusy, and C. C. Standley (Eds.), The Use of Non-Human Primates in Research on Human Reproduction, pp. 322-361. WHO Research and Training Centre on Human Reproduction, Kartolinska Institutet, Stockholm. Michael, R. P., Bonsall, R. W., & Zumpe, D. (1984).The behavioral thresholds of testosterone in castrated male rhesus monkeys (Macaca mubra). Horm. Behav. 18, 161-176. Michael, R. P., Setchell, K. D. R., and Plant, T. M. (1974). Diurnal changes in plasma testosterone and studies on plasma corticosteroids in non-anaesthetized male rhesus monkeys (Macacn mulutta). J. Endocrinol. 63, 325-335. Michael, R. P., and Wilson, M. (1974). Effects of castration and hormone replacement in fully adult male rhesus monkeys (Macaw mularta). Endocrinology 5, 150-159. Michael, R. P., Wilson, M., and Plant, T. M. (1973). Sexual behaviour of male primates and the role of testosterone. In R. P. Michael, and J. H. Crook (Eds.), Comparative Ecology and Behaviour of Primates, pp. 235-313. Academic Press, New York. Mukku, V. R., Murty, G. S. R. C., Srinath, B. R., Ramasharma, K., Kotagi, S. G., and Moudgal, N. R. (1981). Regulation of testosterone rhythmicity by gonadotropins in bonnet monkeys (Mucaca rudiutu). Biol. Reprod. 24, 814-819. Phoenix, C. H. (1974). Effects of dihydrotestosterone on sexual behavior of castrated male rhesus monkeys. Physiol. Behav. 12, 1045-1055. Phoenix, C. H., Slob, A. K., and Goy, R. W. (1973). Effects of castration and replacement
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therapy on sexual behavior of adult male rhesuses. J. Comp. Physiol. Psychol. 84, 472-48 1. Plant, T. M. (1981). Time course of concentrations of circulating gonadotropin, prolactin, testosterone and cortisol in adult male rhesus monkeys (Mucaca mulutta) throughout the 24 h light-dark cycle. Biol. Reprod. 25, 244-252. Steiner, R. A., Peterson, A. P., Yu, J. Y. L., Conner, H., Gilbert, M., TerPenning, B., and Bremner, W. J. (1980). Ultradian luteinizing hormone and testosterone rhythms in the adult male monkey, Macaw fascicularis. Endocrinology 107, 1489-1493. Zumpe, D., and Michael, R. P. (1983). A comparison of the behavior of Macaca fasciculuris and Mucaca mulatta in relation to the menstrual cycle. Amer. .I. Primatol. 4, 55-72.
AND
BEHAVIOR
19, 265-277 (1985)
Effects of Testosterone on the Behavior of Male Cynomolgus Monkeys (Macaca fascicularis) DORIS
ZUMPEAND RICHARD P. MICHAEL’
Department of Psychiatry, Emory University School of Medicine, Atlanta, Georgia 30322 and The Georgia Mental Health Institute, Atlanta, Georgia 30306 To determine the threshold doses of testosterone propionate (TP) that cause clear-cut behavioral changes in the sexual behavior of castrated male cynomolgus monkeys, observations were made on three males during successive S-week treatment periods while they received daily subcutaneous doses of 100 pg TP increasing in octaves to 25.6 mg TP. Males were tested with each of the same two ovariectomized, estrogen-treated females (6 pairs, 330 I-hr behavior tests). To mimic the diurnal plasma testosterone rhythm, TP injections were given at 1600hr and blood samples were obtained at 0800hr (141 samples). Male ejaculatory activity increased at the threshold dose of 200 pg TP per day giving plasma testosterone levels of 830 ng/lOO ml, which is in the physiological range of 6001600 ng/lOO ml for intact males. This threshold dose was eight times higher than in rhesus monkeys on a dose per kilogram body weight basis. There was a further marked increase in ejaculatory performance at higher doses (6.4 to 25.6 mg) giving supraphysiologicaf plasma levels of 4000-9000 ng/lOO ml. There were individual differences in the behavioral changes occurring with TP treatment, and the female partner modulated the effects. These findings were generally similar to those obtained with male rhesus monkeys, but certain species differences were noted. Q 1985 Academic Press, Inc.
Much of what is currently known about the effects of androgens on the sexual behavior of male primates has been derived from castration and hormone replacement studies with male rhesus monkeys (A4ucaca muluttu). Castration decreasesboth the ability to intromit and the frequency of ejaculations; although individual males may continue ejaculating for more than 2 years after surgery. Replacement treatments with high doses of testosterone propionate (2-10 mg/day) restore male sexual activity in a 6- to IO-week period (Michael, 1972; Michael, Wilson, and Plant, 1973; Phoenix, Slob, and Goy, 1973; Phoenix, 1974; Michael and Wilson, 1974). More recently, we reported that very small doses of testosterone propionate (50-100 &day), which gave plasma testosterone levels in the 200-450 ’ To whom correspondence should be addressed: Department of Psychiatry, Emory University School of Medicine, Atlanta, Ga. 30322. 265 0018-506X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
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r&100 ml range, also increased the sexual activity of male castrates (Michael, Bonsall, and Zumpe, 1984). These plasma testosterone levels were below the physiological range for intact males in our laboratory. Individual differences between males and between females, as well as the hormonal status of the latter, exerted powerful modulating effects on the behavioral responses to testosterone treatment. To our knowledge, no systematic castration and hormone replacement studies have been conducted with any male anthropoid primate other than the rhesus monkey, and the present study was undertaken to begin to fill this gap using the closely related cynomolgus monkey (M. fuscicularis). The cynomolgus monkey has interest for several reasons. It is being increasingly used to replace the rhesus monkey in studies on the hormonal control of behavior now that young feral-reared rhesus monkeys are so difficult to obtain. It is taxonomically close to the rhesus but shows some significant differences; it does not breed seasonally (Dang, 1977; Kavanagh and Laursen, 1984), it is less sexually dimorphic, and its copulatory behavior consists primarily of single mount ejaculations (Zumpe and Michael, 1983). To facilitate comparisons with the rhesus monkey, in the present study we administered increasing amounts of testosterone propionate subcutaneously to castrated male cynomolgus monkeys, beginning at doses of 100 pg/day and increasing in octaves to doses of 25.6 mg/day. Behavioral interactions with estrogen-treated females were conducted on a regular basis and plasma testosterone levels were measured by radioimmunoassay to permit correlations with sexual behavior. METHODS Animals. Four male (weighing 5.0-5.2 kg) and two female (weighing 3.1-4.7 kg) cynomolgus monkeys were obtained as adults through dealers directly from Malaysia. Males were castrated 3 months before the start of this experiment, and females had been ovariectomized over 1 year earlier. Unfortunately, one male died during the early months of this study and these results were discarded. Operative techniques were identical to those described in detail elsewhere for rhesus monkeys (Michael and Wilson, 1974). Each male received a pair of Silastic testicular prostheses after castration to restore the appearance and stimulus properties of the scrotal sac. Animals were maintained throughout in individual cages together in a room with windows so that the artificial lighting was supplemented with natural daylight. Temperature was maintained between 20 and 24°C. Food consisted of Purina monkey chow with vitamin supplements and water was available ad libitum. Hormone treatments. Following a lO-week period of testing (two periods each of 5 weeks) to establish behavioral baselines, all males received successive 5-week periods of treatment with testosterone propionate (TP) subcutaneously in 0.2 ml oil at the following dose levels: 100, 200, 400,
TESTOSTERONE
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267
and 800 pg, 1.6, 3.2, 6.4, 12.8, and 25.6 mg per day. All injections were given at 1600 hr, and the first injection of each new treatment was given on the afternoon (Friday) after the last behavioral test of the preceding treatment, so that three injections were given before the first behavioral test of the new treatment (Monday). Beginning 3 months before the start of the study, females received daily subcutaneous injections of 5 pg estradiol benzoate in 0.2 ml oil at 0800 hr. Plasma samples and hormone assays. Starting 3 weeks before the first hormone injection, 3 ml blood was obtained once a week at 0800 hr from the saphenous veins of the untranquilized males that had been previously adapted to the procedure (141 samples). Results were compared with plasma testosterone levels in three 0800 hr blood samples obtained from each male several weeks before castration (9 samples). All samples from a given male were initially analyzed simultaneously. However, owing to the unusually high hormone levels reached in this study, samples from the three highest treatment regimes were reanalyzed at higher dilutions to obtain more accurate estimates. Plasma testosterone levels were determined by radioimmunoassay on 40 ~1 plasma (Bonsall, Baumgardner, and Michael, 1976) using a high-affinity antiserum to testosterone-3-(C)carboxymethyl)oxime-bovine serum albumin produced in these laboratories. The only major cross reaction was with dihydrotestosterone (40%), and this elevated testosterone estimates in plasma samples by up to 16%. The coefficients of variation between duplicates were 7.4% (within assays) and 12.7% (between assays). Water blanks read 3.5 pg and the blank reading was not subtracted. Behavioral testing. Observations were made on oppositely sexed pairs of animals in quiet, isolated rooms from behind one-way vision mirrors in special observation cages (1.19 m wide by 1.07 m deep by 1.14 m high) into which first the male and then the female was introduced at the start of each test session. Tests were of 60 min duration and were conducted 3 days a week (Monday, Wednesday, and Friday). On each test day, two males were each tested with one of the two females, and pairings alternated so that each of the six pairs was tested once a week. No animal was tested more than once per test day. Both tests on a given day were conducted at the same time between 0900 and 1200 hr. The findings reported here are therefore based on a total of 330 behavior tests with six pairs of animals involving three males and two females. Definitions and terminology. The basic descriptions of the behavior observed during tests have been given elsewhere (Zumpe and Michael, 1983). Twenty-nine of the behavioral measures routinely scored were analyzed statistically, but results for only 12 of these are given here: (a) number of ejaculations per test (potency)-the characteristic behavioral response, typical of ejaculation in intact males, performed in coitu while mounting the female; (b) time to first ejaculation-time in seconds from
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the start of the test to the first ejaculation (a default value of 3600 was given where ejaculation did not occur); (c) number of mounts per test; (d) number of male mounting attempts per test-sum of male initiated mounts and attempts to mount that were refused by the female; (e) first mounting attempt latency-time in seconds from the start of the test to the first male mounting attempt (a default value of 3600 was given where there were no mounting attempts); (f) number of intromitted thrusts per test; (g) number of unintromitted thrusts per test; (h) total thrusts per test-sum of intromitted and unintromitted thrusts per test; (i) percentage intromitted thrusts-intromitted thrusts expressed as a percentage of total thrusts per test; (j) male grooming time-time in seconds spent by the male in grooming the female; (k) female grooming time-time in seconds spent by the female in grooming the male; (1) number of male yawns per test (Goy and Resko, 1972). Statistical treatment of results. The significance of differences between plasma testosterone levels during pretreatment and during the different testosterone treatments was assessedby a three-way analysis of variance for repeated measures using a treatment by sample by male design. For statistical purposes only, a male’s treatment mean was used in place of a missing sample value to fill a cell (9 of the 150 samples were not available). Differences between behavioral frequencies were analyzed using a treatment by female by male design, and for this purpose the lo-week pretreatment period was divided into two 5-week periods (Bl and B2). Individual treatment means were compared using the Scheffe test. The significance of associations between changes in plasma testosterone and in behavioral measures was tested by Spearman rank correlation coefficient. RESULTS Testosterone and Male Sexual Activity-Group
Results
The effects of increasing doses of TP on the plasma testosterone and sexual behavior of castrated male cynomolgus monkeys are shown in Table 1. There were highly significant changes in mean plasma testosterone levels at 0800 hr (16 hr after injection), which ranged between 414 and 9129 ng/lOO ml over the 100 pg to 25.6 mg dose range. Physiological levels in these three males when intact were between 600 and 1600 ng/ 100 ml at 0800 hr. Similar levels to these were produced in castrates by doses of 200 pg and 400 ,ug TP per day (Fig. 1). As plasma hormone levels increased, there were highly significant increases in ejaculatory performance. At the 200 pg TP dose, when plasma testosterone reached physiological levels, numbers of ejaculations increased abruptly while ejaculation times decreased equally sharply (Table 1). At the 3.2 mg TP dose level, there appeared to be a further increase in the number of
thrusts
grooming
time
1426 r 95
1085 + 160
3 f 1.4 0.2 2 0.08
3 2 1.4
1388 t
70
1197 + 134
6 f; 2.2 0.2 5 0.08
9 % 4.2
117
IOOS *
120
71 2 5.4 1.6 r 0.49
12.5
and 30 behavior
tests.
2044 c 92
820 + 121
92 k 3.0 5.0 + 0.98
100 + 13.6
6 + 1.9
34 zt 12.4 85 *
248 ? 11% 106 -c 13.5
II L 4.1 I20 zc 16.4
8.0 + 1.17
756 k 244 9.1 f 1.25
953 t 246 8.8 k 1.26 7.8 2 1.00
2464 k 144 1.7 f 0.20
1437 2 98 1.6 + 0.19
8~ ~g TP
Activity
O.%
9.4
122 2206 + 106
807 f
82 5 5.3 5.2 + 1.09
75 f
12 2 4.7
399 _c 201 87 + 11.2
6.0 f
1275 f 216 6.7 + 0.91
3385 ? 263 1.5 z 0.20
1.6 mg TP
TABLE 1 Levels and Sexual
400 fa TP
Testosterone
2074 ? 91
samples
1%3 + 136
1216 -c 174
66 k 4.8 0.7 + 0.29
69 2 8.6
37 e 6.9
18 * 9.7 107 2 9.8
5.2 2 0.56
81s 2 215 6.6 _f 0.67
830 * 113 1.5 + 0.13
200 fig TP
on the Plasma
of P-15 plasma
1700 z 84
1376 *
17 + 4.8 0.2 ? 0.09
20 + 5.4
76 2 10.9
84 k 13.2
0.60
701 2 256 % 2 12.4
3.8 f
857 + 281 94 t 14.8
4.1 2 0.66
2310 c 295 4.7 + 0.55
2637 + 254 5.0 + 0.75
100 pg TP
414 2 50 0.6 + 0.14
2
54 % 4 0.4 -t 0.11
Baseline
Propionate
* Fq.18 for plasma testosterone and F10.m for behavior. ’ Values in the bcdy of the Table are the means * SEM
(sed
bea Female
per test Male yawns per test Male grooming time
test % Intromitted
per
114 + 15.3
thrusts
per test Intromitted
6.4 + 0.92
380 k 183 117 + 15.3
attempt
per
0.09
1
Doses of Testosterone
3170 -c 192 7.3 _f 0.86
0.2 t
Baseline
of Increasing
latency (set) Total thrusts per test Unintromitted thrusts
test Fit mounting
(sea Mount.9 per test Mountiog attempts
cwmo ml) Ejaculations per test Time of ejaculation
Plasma testosterone
Effects
0.72
2264 I?: 133
2504 f
118
635 + 114
764 + 126
85 -c 8.1
2.7
89 + 2.9 5.5 k 1.19
10.1
10 f
246 * 166 95 2 9.2
6.3 2 0.91
68 * 5.9 6.8 -’ 1.22
72 z
28 -+ 6.4
248 t 166 101 5 12.0
5.0 t
139 2 87 7.2 2 0.74
548 -t 193 6.8 -t 0.92
6.4 mg TP
Cynomolgus
4149 k 278 2.6 t 0.14
Male
3712 + 243 2.2 2 0.19
3.2 mg TP
of Castrated
166 9.5
105 2458 z. 115
633 k
a7 2 3.3 4.0 + 0.87
90 e 8.2
1s 2 4.7
248 t 105 f
5.2 k 0.66
51 + 20 7.5 A 0.59
6 Pairs)”
5.2
8.1
844 ‘c 116 2191 + 110
45.4 3.4
6.0 89 ” 3.8 5.8 -+ 1.23
9.3
7.1
7 Et 1.8 86 t
1.3 0.5
126 5 120 93 2 9.6
2.3
11.7 1.9
134 + 120 6.7 -t 0.68 4.9 k 0.59
44.6 19.0
F*
9129 2 743 2.6 + 0.19
25.6mgTP
(3 Males.
5032 k 253 3.2 ? 0.14
12.8mgTP
Monkeys
co.001 <0.025
NS NS
NS
~0.001
P
270
! ! -I’1---_ ..-
ZUMPE AND MICHAEL
61 62
400~ b6m9 64mq 25.61rq 200~0 600~ 32mg I2.6mg
TESTOSTERONE PROPIONATE DOSAGE FIG. 1. Changes in ejaculations and plasma testosterone levels in three castrated male cynomolgus monkeys (6 pairs) given increasing daily doses of testosterone propionate (TP) subcutaneously. Data from 330 mating tests and 141plasma samples. In this and subsequent figures Bl and B2 = two successive S-week periods of pretreatment baseline. Vertical bars given standard errors of means. Interrupted horizontal lines indicate the upper and lower limits of the physiological plasma testosterone range for these males when intact.
ejaculations, and this was associated with plasma levels greatly in excess of the physiological range. Mounts, mounting attempts and mounting attempt latencies did not change significantly and no consistent patterns were observed, perhaps because these behaviors had not yet been affected by castration. However, intromitted thrusts increased significantly and unintromitted thrusts decreased significantly at 200 pg TP but did not change further at higher doses. Percentage intromitted thrusts increased significantly during the 200 pg TP treatment and reached a maximum with the 800 pg TP treatment. The numbers of male yawns, an androgendependent behavior in rhesus monkeys (Goy and Resko, 1972) increased markedly at the 800 pg TP dose. This increase reached statistical significance at the 1.6 mg TP dose but showed no further changes at higher dose levels. There were no significant effects of testosterone on measures of agonistic and social behavior, except for decreases and increases in male and female grooming times, respectively (Table 1). There was a positive association between changes in plasma testosterone and changes in ejaculations (rS = 0.93, N = 10, P < 0.001) and a negative association between plasma testosterone and times of ejaculation (r, = -0.90, N = 10, P < 0.001). Changes in plasma testosterone were also significantly associated with changes in intromitted thrusts, unintromitted thrusts, percentage intromitted thrusts, yawns, and male and female grooming times (r, = kO.70 to kO.85, N = 10, P < 0.0.5-0.01). There
TESTOSTERONE
AND BEHAVIOR
271
were no significant associations between changes in plasma testosterone and changes in total thrusts, mounts, mounting attempts or mounting attempt latencies (Y, = +0.21 to kO.47, N = 10, NS). Thus, ejaculatory performance was more closely related than any other behavioral measure to plasma hormone levels, including those reflecting the ability to obtain intromission. Testosterone and Male Sexual Activity-Individual Males Although plasma testosterone levels in the different males were generally rather similar at each dose level (F2,36= 0.57, NS), there were highly significant differences between males for all behavioral measures (F2,r0 = 5.8-209.0, P < 0.025-0.001) except percentage intromitted thrusts and male grooming times. Male 2 made far fewer mounting attempts and yawns (Fig. 2) and fewer intromitted and unintromitted thrusts (Fig. 3) in response to testosterone than either of the other two males. There were also individual differences in the behavioral response patterns with increasing levels of plasma testosterone. Males 1 and 2 showed a fairly sharp increase in numbers of ejaculations at the 200 ,ug TP dose. Ejaculations increased again with higher doses to reach maximal levels during the 12.8 mg TP treatment. Male 3 also showed a definite increase in ejaculations at 200 pg TP, but in this male ejaculations appeared to increase progressively over the entire dose range (Fig. 2). Individual differences were also apparent in numbers of intromitted thrusts and percentage intromitted thrusts (Fig. 3): male 1 showed a graded increase with dose which reached a plateau at the 800 pg TP level, whereas males 2 and 3 showed a sharp response at a threshold dose of 200 pg TP. It was of interest that male 3 showed a very abrupt increase in percentage intromitted thrusts at 200 pg TP but a graded increase in ejaculations over the whole dose range. Testosterone and Male Sexual Activity-Individual Females Both female partners were ovariectomized and treated with 5 pg estradiol benzoate per day. While there were no significant behavioral differences between them (F,,Z = 0.2-13.6, NS), there was nevertheless evidence that they modulated the behavioral effects of treating males with testosterone. Figure 4 shows the mean numbers of ejaculations and percentage intromitted thrusts received by each female from all males. With female A, there appeared to be a close relationship between changes in the two behavioral variables; percentage intromitted thrusts and ejaculations increased markedly at the 200 pg TP dose and tended to covary at higher doses (Fig. 4, left). With Female B, on the other hand, there was no increase in ejaculations between the 200 pg and 1.6 mg. TP doses although the percentage of intromitted thrusts increased markedly as they did with female A; this produced a clear dissociation between the two behavioral
272
ZUMPE AND MICHAEL
TESTOSTERONE
PROPIONATE
DOSAGE
FIG. 2. Comparison of changes in the sexual behavior and plasma testosterone levels of three castrated male cynomolgus monkeys given increasing daily doses of TP. There were broadly similar changes in ejaculations but clear individual differences in the levels of other behavioral indices. In this and Fig. 3, the data for each male were from 110 mating tests and 41-43 plasma samples. Symbols as in Fig. 1.
variables (Fig. 4, right). Some property of female B must have been responsible for this. DISCUSSION These findings were quite clear-cut and reached acceptable levels of statistical significance using analyses of variance, so that it is permissible to make some comparisons with data from similarly treated male rhesus monkeys (Michael et al., 1984). Ejaculatory activity increased in castrated male cynomolgus monkeys with 200 E.L~TP per day which gave plasma levels of testosterone in the physiological range for intact males. In rhesus
273
TESTOSTERONE AND BEHAVIOR MALE
I
MALE
2
MALE
3
c
IO0
50
0
TESTOSTERONE
PROPIONATE
DOSAGE
FIG. 3. Comparison of changes in three measures of thrusting activity and in plasma testosterone levels of three castrated male cynomolgus monkeys given increasingly daily doses of TP. Although there were marked individual differences, in all three males intromitted thrusts reached maximum levels at 800 pg TP, while unintromitted thrusts declined to very low levels at this dosage. The ability to obtain intromission (middle panel) was fully restored with the 800 pg TP treatment. Symbols as in Fig. 1.
monkeys, ejaculatory activity increased with 50 pg TP per day which gave plasma levels of testosterone well below the physiological range for this species. It should be remembered, of course, that male rhesus monkeys are about twice the body weight of male cynomolgus monkeys, and so the rhesus monkey may be regarded as considerably (eight times) more sensitive to testosterone with respect to ejaculation than the cynomolgus monkey. This difference may relate to the fact that the rhesus monkey, unlike the cynomolgus monkey (Mahone and Dukelow, 1979), is a seasonal breeder which undergoes testicular regression during the
274
ZUMPE AND MICHAEL FEMALE
a $ oI
A
FEMALE
B
T/i
;/‘,/‘l o--Q ) , ) , , 81 IoOpg 4C9q 82
, , , , , ,
L
Wnq 6.4mg ZbBmq 2ooJq BooJIg 32mq 12~6rn6
62
TESTOSTERONE
PROPIONATE
200~9 BOOM 32ma 12.6~ DOSAGE
FIG. 4. The identity of the female partner helped to determine the responses of castrated male cynomolgus monkeys to TP administration. With female A, ejaculations and intromitted thrusts were closely associated throughout the dose range. With female B, a dissociation occurred, and the ability to intromit was fully restored at 800 pg TP whereas ejaculations did not increase until 3.2 mg TP. Each female was tested with the same three males. Symbols as in Fig. 1.
nonmating season and is more behaviorally sensitive to very small increases in plasma testosterone when levels are low. However, higher doses (3.212.8 mg) giving plasma testosterone levels greatly in excess of the physiological ranges resulted in further increases in the ejaculatory performance of both cynomolgus and rhesus monkeys. The two species differed in their potency and in their ability to obtain intromission. Cynomolgus monkeys showed a sharp increase in percentage intromitted thrusts (10% increased to 90%) with 800 pg TP per day, while in rhesus monkeys this change was much smaller (0% increased to 25%) with even higher doses. We are not sure if this difference, like the higher potency of the cynomolgus monkey (generally two ejaculations per test instead of one), is a genuine species difference related to its single-mount ejaculatory pattern or due to the longer period in captivity of our rhesus monkeys. Since the intromissions of cynomolgus monkeys were fully restored with 800 pg TP, the increase in ejaculations at much higher doses of testosterone (12.8 mg) could not readily be ascribed to a peripheral action of androgen on the penis. The possibility that higher doses caused an increase in male sexual motivation received some support from the observation that, as dose levels increased from 400 pg to 25.6 mg TP, there was a significant decrease in mean numbers of intromitted thrusts to ejaculation (33.3 r 8.42 to 14.0 + 0.22) (F,o,zo = 5.9, p < 0.001). In this study, the dissociation between changes in intromitted thrusts and
TESTOSTERONE
AND
BEHAVIOR
275
ejaculatory performance was most obvious in tests involving female B (Fig. 4); suggesting that some property of this female was responsible for the effect rather than a hormone-dependent mechanism in the males. It is possible that female A was made fully receptive by 5 Fugestradiol benzoate per day while female B was not, because we have noted a similar dissociation occurring in male rhesus monkeys when they were paired with ovariectomized, untreated females. Hormone injections were given at 1600hr, 16 hr before blood sampling, to mimic as closely as possible the diurnal plasma testosterone rhythm of intact animals (Steiner, Peterson, Yu, Conner, Gilbert, TerPenning, and Bremner, 1980; Michael et al., unpublished observations). The diurnal rhythm of cynomolgus monkeys, like that of rhesus and bonnet monkeys (Michael, Setchell, and Plant, 1973; Plant, 1981; Kholkute, Joseph, Joshi, and Munshi, 1981; Mukku, Murty, Srinath, Ramasharma, Kotagi, and Moudgal, 1981), results in low daytime and high nighttime plasma testosterone levels. The 24-hr plasma testosterone profile following a single injection of testosterone propionate at 1600 hr was similar to that in rhesus monkeys (Michael et al., 1973). In two of the males, injecting 400 pg testosterone propionate at 1600 hr resulted in mean plasma testosterone levels of 1263, 864, 675, 650, and 607 ng/lOO ml at 2, 6, 16, 20, and 24 hr after injection. Testosterone levels in 0800 hr samples in our animals when they were intact ranged between 600 and 1600 r&100 ml, which is consistent with a morning mean of 9.1 rig/ml in another study (Steiner et al., 1980). Over the 1.6-12.8 mg TP dose range, plasma testosterone levels increased only modestly from about 3400 ng/lOO ml to about 5000 ng/lOO ml, perhaps because of changes in metabolism and clearance; but levels doubled to about 9000 ng/lOO ml with the 25.6 mg TP treatment. Adjusting for body weight differences, the cynomolgus and rhesus monkeys showed quite similar plasma hormone levels over the 100 pg to 12.8 mg dose range. These findings extend our knowledge of the effects of testosterone on behavior to another, closely related species of anthropoid primate. On a dose per kilogram body weight basis, the threshold dose of TP for ejaculation was eight times greater in the cynomolgus monkey than in the rhesus monkey. It should be noted, however, that “threshold dose” is not an absolute constant but may vary with time since castration, with treatment duration, with increasing or with decreasing doses, with return to baselines between doses, and with other differences in experimental design. In both species, ejaculatory activity increased progressively with increasing plasma testosterone levels, but effects were modulated by the identity of the female partner. Clear behavioral changes were observed over the physiological dose range, and in both species additional wellmarked increases in ejaculatory activity occurred when plasma testosterone reached supraphysiological levels. However, cynomolgus monkeys, unlike
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rhesus monkeys, showed no increase in ejaculatory performance when plasma hormone levels were below the physiological range, and their ability to intromit was fully restored at doses of TP well below those producing maximum ejaculatory performance. The male cynomolgus monkey offers considerable promise for studies on the hormonal control of behavior. ACKNOWLEDGMENTS This work was supported by USPHS Grant MH 19506, and general research support was provided by the Georgia Department of Human Resources. Both are gratefully acknowledged.
REFERENCES Bonsall, R. W., Baumgardner, D. G., and Michael, R. P. (1976). A computerized semiautomated radioimmunoassay for plasma testosterone. J. Steroid Eiochem, 7, KU858. Dang, D. C. (1977). Absence of seasonal variation in the day length of the menstrual cycle and fertility of the crab-eating macaque (Macaca fuscicularis) raised under natural daylight ratio. Ann. Biol. Anim. Biochim. Biophys. 17, 1-7. Goy, R. W., and Resko, J. A. (1972). Gonadal hormones and behavior of normal and pseudohermaphroditic nonhuman female primates. Rec. Prog. Horm. Res. 28, 707733. Kavanagh, M., and Laursen, E. (1984). Breeding seasonality among long-tailed macaques, Macaca fascicularis, in Peninsular Malaysia. Int. J. Primatol. 5, 17-29. Kholkute, S. D., Joseph, R., Joshi, U. M., and Munshi, S. R. (1981). Diurnal variations of serum testosterone levels in the male bonnet monkey (Macaca radiara). Primates 22, 427-430. Mahone, J. P., and Dukelow, R. W. (1979). Seasonal variation of reproductive parameters in the laboratory-housed male cynomolgus macaque (Macaca fuscicularis). J. Med. Primatol. 8, 179-183. Michael, R. P. (1972). Determinants of primate reproductive behaviour. In E. Diczfalusy, and C. C. Standley (Eds.), The Use of Non-Human Primates in Research on Human Reproduction, pp. 322-361. WHO Research and Training Centre on Human Reproduction, Kartolinska Institutet, Stockholm. Michael, R. P., Bonsall, R. W., & Zumpe, D. (1984).The behavioral thresholds of testosterone in castrated male rhesus monkeys (Macaca mubra). Horm. Behav. 18, 161-176. Michael, R. P., Setchell, K. D. R., and Plant, T. M. (1974). Diurnal changes in plasma testosterone and studies on plasma corticosteroids in non-anaesthetized male rhesus monkeys (Macacn mulutta). J. Endocrinol. 63, 325-335. Michael, R. P., and Wilson, M. (1974). Effects of castration and hormone replacement in fully adult male rhesus monkeys (Macaw mularta). Endocrinology 5, 150-159. Michael, R. P., Wilson, M., and Plant, T. M. (1973). Sexual behaviour of male primates and the role of testosterone. In R. P. Michael, and J. H. Crook (Eds.), Comparative Ecology and Behaviour of Primates, pp. 235-313. Academic Press, New York. Mukku, V. R., Murty, G. S. R. C., Srinath, B. R., Ramasharma, K., Kotagi, S. G., and Moudgal, N. R. (1981). Regulation of testosterone rhythmicity by gonadotropins in bonnet monkeys (Mucaca rudiutu). Biol. Reprod. 24, 814-819. Phoenix, C. H. (1974). Effects of dihydrotestosterone on sexual behavior of castrated male rhesus monkeys. Physiol. Behav. 12, 1045-1055. Phoenix, C. H., Slob, A. K., and Goy, R. W. (1973). Effects of castration and replacement
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therapy on sexual behavior of adult male rhesuses. J. Comp. Physiol. Psychol. 84, 472-48 1. Plant, T. M. (1981). Time course of concentrations of circulating gonadotropin, prolactin, testosterone and cortisol in adult male rhesus monkeys (Mucaca mulutta) throughout the 24 h light-dark cycle. Biol. Reprod. 25, 244-252. Steiner, R. A., Peterson, A. P., Yu, J. Y. L., Conner, H., Gilbert, M., TerPenning, B., and Bremner, W. J. (1980). Ultradian luteinizing hormone and testosterone rhythms in the adult male monkey, Macaw fascicularis. Endocrinology 107, 1489-1493. Zumpe, D., and Michael, R. P. (1983). A comparison of the behavior of Macaca fasciculuris and Mucaca mulatta in relation to the menstrual cycle. Amer. .I. Primatol. 4, 55-72.