Social influences on circulating levels of cortisol and prolactin in male talapoin monkeys

Physiology &Behavior,Vol. 30, pp. 361-369. Pergamon Press Ltd., 1983. Printed in the U.S.A.

Social Influences on Circulating Levels of Cortisol and Prolactin in Male Talapoin Monkeys J. A . E B E R H A R T , 1 E. B. K E V E R N E

A N D R. E. M E L L E R

Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, England R e c e i v e d 28 A u g u s t 1982 EBERHART, J. A., E. B. KEVERNE AND R. E. MELLER. Social influences on circulating levels of cortisol and prolactin in male talapoin monkeys. PHYSIOL BEHAV 30(3) 361-369, 1983.--Three captive groups of adult talapoin monkeys (Miopithecus talapoin), each with four males and four or five ovariectomized, estrogen-treated females, were observed. Behavioral interactions were recorded and levels of cortisol and prolactin were measured. In each group, males formed a linear dominance order, determined by the direction of spontaneous aggression; highest male rank was associated with frequent socio-sexual interactions and lowest rank with infrequent socio-sexual behavior. The first study compared males' cortisol and prolactin titers when all males were either housed with estrogen-treated females, or singly caged. The two lowest-ranking males of each group had elevated cortisol levels when group-housed; prolactin levels did not reflect these changes. In the second study, males of one group interacted with females when only the lowest-ranking, or the highest-rankiog, female was made attractive (i.e., received estradiol), while the other females were present, but not estradiol-treated. Across these conditions (1) frequencies of some socio-sexual and aggressive behaviors changed, (2) cortisol levels increased in all males, and (3) prolactin levels decreased in three of four males. Dissociation of changes in cortisol and prolactin titers suggests that these hormones may be differentially responsive to social modifications, not simply reflecting a single intervening variable, such as "stress." Talapoin monkeys Primates

Sexual behavior

Aggression

A L T H O U G H the greatest number of endocrine studies with primate social groups have focused on testosterone (reviewed in [3,7]), there are numerous reports concerning adrenocortical activity. Among these are studies using immature subjects [6, 14, 28, 29], short-lived groups [5,30], indirect measures of adrenocortical activity [15, 20, 22], and the introduction of potentially traumatic stimuli [21,32]. Most of these studies have interpreted differences, or changes, in measures of adrenocortical activity to reflect differing levels of " s t r e s s . " However, what is meant by " s t r e s s " is rarely made clear. One study [25] has described effects of social stimuli on plasma cortisol levels in adult male squirrel monkeys, but data from lowest-ranking males in this investigation were not fully analyzed. Recently, a study with social groups o f talapoin monkeys [34] suggested that serum cortisol levels in lower-ranking males may be more responsive to changes in the social environment than those of highranking males. Consequently, the effects of social influences on the cortisol titers of all males in a primate social group deserve further attention. Prolactin has been measured in pharmacological experiments using rhesus [27], mangabey, patas [1], and talapoin [23] monkeys, and in a study concerning the effects of bleeding procedures on hormone levels in rhesus monkeys [26].

Dominance

Cortisol

Prolactin

Stress

As with corticosteroids, elevated prolactin levels have most often been assumed to reflect increased " s t r e s s . " In contrast to adrenal and gonadal hormones, though, prolactin has infrequently been considered in endocrine studies of primate social behavior. Experiments with captive talapoin monkeys suggest that social interaction with females may influence serum prolactin levels in both males and females [16,34], particularly in the lowest-ranking animals [4,11]. However, because these experiments involved a limited number of social conditions, further work is needed to assess the generality of these findings. If it is true that circulating titers of cortisol and prolactin primarily reflect levels of a single intervening variable, such as " s t r e s s , " then the pattern of changes in levels of these hormones should be similar. This report considers social influences on cortisol and prolactin levels of males in three groups of male and female talapoin monkeys. The first experiment describes hormone levels when males were isolated and when all males of each group had access to ovariectomized, estrogen-treated females. A second study involved estrogen treatment of selected females in one social group; this study describes changes in males' behavioral interactions and in their serum levels of cortisol and prolactin. These studies address the relationship between social in-

~Requests for reprints should be addressed to J. A. Eberhart, Rockefeller University, 1230 York Avenue, New York, NY 10021.

Copyright © 1983 Pergamon Press Ltd.--0031-9384/83/030361-09503.00

362

EBERHART. KEVERNE AND MELLt£R

teractions and male endocrine state and the lack of correspondence between levels of cortisol and prolactin in these males. GENERAL METHOD

Animals Over a four-year period nine adult female, and twelve adult male, talapoin monkeys (Miopithecus talapoin) were used. All animals had lived in the laboratory at least six months prior to these studies; except for two males (Fo and Da) reared by Dr. T. E. Rowell, all monkeys were wild-born. Males weighed between 1200 and 2500 g, and females between 800 and 1500 g. All males but two were intact; throughout this investigation, castrated males (C1 and C2) received testosterone in the form of subcutaneous silastic implants maintaining plasma levels of 4-8 ng/ml [12]. Each female had been ovariectomized at least three years previously, and each received a subcutaneous implant of estradiol-17fl at intervals during these studies. Estradiol implants fully developed the sex-skin swelling of each female and maintained plasma estradiol levels approximating the normal range for the follicular phase of the talapoin menstrual cycle [16]. Table 1 indicates the composition of social groups. In each social group dominance rank was assigned to males according to the direction of aggression (attacks and threats) among males of that group. (I.e., the highest-ranking male gave aggression to other males, but received little or none in return; conversely, the lowestranking male directed little aggression to other males, but received much aggression from them.)

Caging Each social group lived in a large (approximately 3.5 x 1.5 x 1.7 m) indoor cage occupying a separate room and isolating group members from direct contact with other monkeys. These cages were essentially identical, containing perches, food hoppers, and an ad lib water supply and were easily divided into three compartments of equal size. (Further details appear in [19].) When temporarily removed from the group cage during experimental manipulations (described below), animals were not observed, but were housed singly in small cages (018x0.8x0.5 m) in a separate room. Animals in small cages had visual, acoustic, and olfactory contact with other talapoin monkeys in that room. In all rooms the temperature was maintained at 21-27°C, and fluorescent illumination provided from 08:00-20:00. Once daily monkeys received fresh fruit, mixed seeds, and Mazuri Primate Diet (British Petroleum Plc.).

Procedures On each day of data collection an observer behind oneway glass recorded behavioral interactions among all members of a social group during two 50-minute periods (beginning at 08:00 or 09:00, and 17:00). This method of observation is described elsewhere [2] as "focal group sampling": all monkeys in the cage constitute the focal group, with the observer recording all occurrences of selected behaviors (below) as "events." For Groups A and B, each treatment represents ten 100-rain observations (1000 rain) over a 4-week period; for Group C, each treatment represents a minimum of sixteen 100-rain observations (1600 min) over a 4-week period. Inter-observer reliability was greater than 80% for

aggression and greater than 90% for socio-sexual behavior. Animals of Group A had continuous access to all sections of their group cage, and therefore, to each other. In Groups B and C, however, males were confined to two-thirds of their group cages, while females were confined to the remaining third. Only during observation was a partition removed, allowing contact between males and females. When partitions were in place males and females could hear, see. and possibly smell each other through cracks between panels of the cage, but they could not directly interact. Immediately following the 17:00 observation period on alternate observation days 2-4 ml of blood was taken by femoral venipuncture from each ketamine- (Vetalar. ParkeDavis, 0.2 ml) anesthetized male. Animals were routinely bled twice weekly and were habituated to capture. Males of each group were caught and bled in a fixed order, a procedure requiring less than 30 min, typically less than 15 min. In captive talapoin monkeys unaccustomed to capture, a bleeding procedure more disruptive than that used here had no significant effect on levels of cortisol or prolactin over this interval [10]. Plasma (Groups A and B) or sera (Group C) were separated and stored at -20°C until assay by radioimmunological procedures described elsewhere (prolactin. and cortisol for Group C: [34]; cortisol for Groups A and B: [18]). Behavioral data were not collected on the day following blood sampling.

Behaviors During each observation session, all animals in a group were visible, and were monitored, continuously; instances of aggressive and socio-sexual interactions (below) were recorded, indicating the actor and recipient of each behavior. The behaviors recorded are described in detail elsewhere [9]. Briefly, socio-sexual interactions include presentations (solicitations) by females to males, investigations of females' sex skin by males, mounts of females by males, and ejaculatory mounts of females. Aggression includes attacks (chase, grab, bite), threats (threat face, open-mouth threat, lungeL and displacements (pushing another monkey aside and taking its place, or simply assuming its position). The response of animals to aggression (i.e., ignore, freeze, withdraw) were also recorded.

Statistical Analysis Endocrine data were analyzed with parametric statistics appropriate for independent samples [24]. Behavioral data, not assumed to be normally distributed, were analyzed with nonparametric statistics for independent samples [31]. Neither endocrine nor behavioral data from social groups of monkeys are logically independent, but the data better fit a statistical model assuming independence of observations than one assuming relatedness--i.e., consistency of unique characteristics over time (see [31,33]). No effects of season have been found for either behavioral or endocrine variables in laboratory-housed talapoin monkeys (Keverne and Meller, unpublished data); therefore, season was disregarded in experimental design and data analysis. EXPERIMENT 1 One purpose of this study was to summarize socio-sexual and aggressive interactions of male talapoin monkeys housed in social groups with estrogen-treated females. The second purpose was to describe circulating levels of cortisol and

CORTISOL A N D P R O L A C T I N IN M O N K E Y S

363

TABLE 1 COMPOSITION OF TALAPOIN SOCIAL GROUPS Group A Males

Females

FI F2 F3 F6 F7 (Group formed 12/74) M

C1 C2 330

Group B Males Fo Da Jf Sh

Group C

Females

Males

F12 F14 F9 F8

S D I N

(Group formed 2/76)

weeks, beginning more than a week after being placed in isolation. Male 330 died after the Group condition. Therefore, data for this male represent blood samples taken during a period o f isolation before the Group condition.

Females

RESULTS Dominance Rank

F1 F2 F3 F7

Data used to assign male rank in Groups A and C during this period appear elsewhere [12]. In Groups A and B rank order was stable throughout this study: (Group A) M > C 1 > C 2 > 3 3 0 , (Group B) F o > D a > J f > S h . In Group C the hierarchy during the Group period was S > I > D > N ; however, shortly after this time (and before isolation) the hierarchy changed to S > D > I > N , remaining stable in this configuration for several years. F o r clarity, all figures identify males by both name and rank.

(Group formed 4/78)

All males except C1 and C2 were intact, and all females were ovariectomized.

Socio-sexual Behavior

prolactin in these males after they were removed from the groups and housed in isolation. The third purpose was to compare cortisol and prolactin levels during isolation with levels in the social groups. In this way it may be possible to determine the potential of social stimuli to influence circulating levels of these hormones in group-living primates. METHOD Behavioral and endocrine data were collected as described in the General Method section. In the Group condition all males of each social group lived in a large cage with all (estrogen-treated) females of that group (Table 1). During Isolation each male lived alone in a small cage, having no direct contact with other monkeys. With the exception o f one male (330), Isolation data represent weekly or biweekly blood samples taken over a continuous period of at least four

A detailed analysis of socio-sexual interactions for the males of Groups A and C during the Group period appears elsewhere [12]. The socio-sexual behaviors of males in all three groups are briefly described here to illustrate the rankrelated differences in these behaviors, differences that may be associated with levels of cortisol and prolactin in males. Table 2 presents a summary o f males' behavioral interactions during the Group period. In each group the highestranking male was sexually most active, and the lowestranking male was considerably less active. In Groups A and C the highest-ranking male not only received far more presentations from females than did other males, but he was also the only male to regularly mount and ejaculate with females. Lower-ranking males in Groups A and C rarely mounted, and never ejaculated with, females. In Group B the second-ranking male (Da) received as many presentations from females, and mounted females as often, as did the

TABLE 2 SUMMARY OF MALES' BEHAVIORALINTERACTIONS DURING THE GROUP PERIOD

Males

Presentations Received from Females

Group A (n= 10) M 22.5 (14.3, 31.0) C1 0 (0, 0.3) C2 * 330 0.5 (0, 3.3) Group B (n=10) Fo 49.5 (42.0, 63.3) Da 52.0 (32.3, 64.0) Jf 0 (0, 1.0) Sh * Group C (n=24) S Ill.0 (97.0, 127.0)

Mount Females

Ejaculatory Mounts

Attack Males

Threaten Males

4.5 (2.0, 7.3) * * *

2.0 (1.0, 3.3) * * *

2.5 (0.8, 4.3) 2.0 (0, 3.8) 0.5 (0, 1.0) •

4.5 (2.8, 6.0) * 0 (0, 0.3) •

12.5 (10.0, 13.8) 9.5 (5.8, 13.3) * *

9.0 (8.0, 9.3) * * *

0 (0, 0.3) 0 (0, 1.5) 0 (0, 0.3) *

* 3.0 (0, 8.5) * * 1.0 (0, 2.0) 1.5 (0, 3.8) 3.0 (1.0, 6.5) 0 (0, 1.0)

9.0 (6.3, 11.0)

1.0 (0, 2.0)

1.5 (0, 5.0)

I

4.0 (3.0, 6.8)

*

*

14.5 (5.3, 21.3)

D N

7.0 (5.3, 9.0) *

* *

* *

26.5 (18.5, 35.0) 0 (0, 1.8)

Values represent Medians (L.Q., U.Q.) per 100 minutes of observation; n---number of 100-minute observations. *Indicates that Median, L.Q., and U.Q. each=0.

364

E B E R H A R T , K E V E R N E A N D MELI,lv:R Levels of cortisol and prolactin durin 9 isolation.

TABLE 3 AGGRESSION RECEIVED BY MALES FROM OTHER MALES AND FROM FEMALES Aggression From Other Males Males Attack Group A (n= 10) M 0 CI 7 C2 9 330 38 Group B (n = 10) Fo 0 Da 0 Jf 25 Sh 5 Group C (n=24) S 15 I

D N

9

28 996

Cortisol Male 1 2 3 Rank Group A

Aggression From Females

Threat

Displace

Attack

Threat

Displace

4 17 11 21

0 8 38 37

0 5 7 4

2 4 5 1

7 13 12 6

Pr01actm 4

I

,7

~

Fs0

!4o), M n: 7

,,,

CI 7

C2

330

9

12

,.o

7

6

5

7 18 16 282

1 7 1 3

0 0 1 0

2 14 30 18

4 13 24 157

5 43 87 17

18 13 20 69

0

27

2

8

8 4

7 6

Fo n~8

~-

o tD

Da 8

Jf 8

Sh 8

8

8

9

if

T

Group B

+

2 1 31 14

4

8

7

Group C

Values represent totals observed during n × 100 minutes of observation over a 4-7 week period. Details of observation given in the text.

8

highest-ranking male (Fo); however, unlike the highestranking m a l e - - w h i c h frequently ejaculated with f e m a l e s - the second-ranking male never ejaculated with females during this period. Thus, among males of each group, copulation (i.e., mounts with ejaculation) was the prerogative of the highest-ranking male.

F.0

0Do

o S n:8

D 8

I 8

N 8

8

8

8

8

FIG. t. Mean levels (+S.D.) of cortisol (at left) and prolactin (at right) during Isolation for all males (rank 1-4) of each social group. Details are given in the text. n=Number of blood samples taken over a period of at least 4 weeks.

Aggression Table 2 presents the median levels of aggression (attacks and threats) directed by the males of each group to other males. By definition (above), higher-ranking males gave aggression to other males, but lowest-ranking males gave little or none. However, only in Group A did the highest-ranking male (M) direct more aggression to other males than did the second- or third-ranking males [12]. In contrast to frequent male-male aggression, males rarely gave aggression to females. Considering attacks, threats, and displacements directed by each male to all females, the median frequency of these behaviors is zero, except in two cases. In Group B, Da attacked females a median (Lower-, U p p e r Quartile) of 0.5 (0, 2.0) times, and in Group C, S threatened females a median of 1.0 (0, 2.0) times, per 100 minutes of observation. The levels of aggression received by each male during the Group period are too low to allow either statistical analysis or presentation of median values. F o r this reason, Table 3 presents, for each male, the total number of attacks, threats, and displacements received from other males and from females. Table 3 indicates that all males received some aggression from both males and females. In each group there is more variabifity across males in the amount o f aggression received from other males than from females. Following the definition of dominance, highest-ranking males received little male aggression, while lower-ranking males (especially males ranked 3 and 4) received relatively high levels. Conse-

quently, a high proportion of the aggression received by higher-ranking males was from females, whereas most of the aggression received by lower-ranking males was from other males.

Hormone Levels During Isolation For the males of each group, Fig. 1 shows mean levels of cortisol and prolactin during isolation. In each group, cortisol levels differed among males, Group A: F(3,31)=4.86, p<0.01; Group B: F(3,28)=26.8, p<0.001; Group C: F(3,28)=8.87, p<0.001; however, there is no pattern of differences common to all groups. Isolation levels of prolactin also differed among the males in each group, A: F(3,23)=8.23, p<0.001; B: F(3,27)=3.23, p<0.05; C: F(3,28)=58.5, p<0.001. In Groups A and C, the highestranking male had higher prolactin levels than did the lowestranking male of the same group, A: t(14)=3.882,p<0.005; B: t(14)=10.652, p<0.0001; in Group B, the opposite is true, t(13)=5.556, p<0.001. In all groups males of intermediate ranks had prolactin levels that were similar to, or interposed between, those of highest- and lowest-ranking males. The pattern o f prolactin titers does not parallel the pattern of cortisol levels.

CORTISOL AND PROLACTIN IN MONKEYS

Male 1 Rank

Cortisol 2

4

B

GroupA

1

365

Prolactin 2 B

t(12)=7.273, p<0.0001, and unchanged in three males (M, S, D). Higher cortisol titers in the two lower-ranking males of each group during the Group period may reflect the frequent aggression received by these males at that time (Table 3). Changes in prolactin levels neither followed the pattern seen in cortisol, nor were consistent across groups. However, in Group C, males ranking one, two, and three each had moderately higher prolactin titers when group-housed (3 comparisons: each, t(20)>3.447, p<0.005), whereas the lowest-ranking male had considerably higher levels during the same period, t(20)=6.875, p<0.0001; Fig. 2).

***

M

C1

C2 330

• I

150

CroupB

! i

DISCUSSION

Rn ~ -50~

GroupC

00~

250.

200150.

10050O.

n

S

i

D

I

N

*p<.01 *'p<'001 ***p<.0001

FIG. 2. Comparison of mean levels of cortisoi (at left) and prolactin (at right) for all males of each social group: Isolation vs. Group periods. Bars above the horizontal axis indicate the percentage by which hormone levels were higher during the (4--7 week) Group period; those below the axis the percentage by which they were lower during this period. Statistical analysis is presented in the text: *p<0.01, **p<0.001, ***p<0.0001 by unpaired t-test (2-tailed).

Hormone Levels in Social Groups To emphasize differences between experimental conditions, hormone data for the Group period are expressed in relative terms. Figure 2 presents hormone levels for each male during the Group period as a percentage of that male's Isolation level. (For example, if there was no difference between conditions, the value is zero; if the Group level was twice the Isolation level, the value is + 100.) Considering differences in mean cortisol levels between treatments, one finding is consistent across groups. In each case, the two lower-ranking males had significantly higher cortisol titers when group-housed than after removal to isolation, C2: t(15)=3.226, p<0.01; 330: t(18)=6.861, p <0.0001; Jf: t(12)=7.897, p<0.0001; Sh: t(13)=10.083, p<0.0001; I and N: both, t(20)>4.794, p<0.0001. By contrast, there is no consistent pattern of changes in cortisol levels for the two higher-ranking males of these groups: Group period cortisol levels were higher in two of these males, CI: t(13)=2.270, p<0.05; Fo: t(12)=2.428, p<0.05, lower in one male, Da:

Although there are differences between groups in the frequencies of males' socio-sexual and aggressive interactions when housed with females, the distribution of these behaviors among males is similar in each group (Tables 2, 3). In agreement with previous reports [8,34], the frequency of males' socio-sexual interactions with females was related to male rank: the highest-ranking male of each group frequently engaged in socio-sexual interactions with females, whereas the lowest-ranking males rarely engaged in these interactions. During isolation, levels of cortisol and prolactin differed among males, but there is no pattern common to all three groups (Fig. 1). Nor is there a consistent association between levels of cortisol and levels of prolactin. Although endocrine differences among isolated males may reflect previous social experience (during the Group period), the differences persist, even after a month of isolation. Therefore, it is not clear whether such differences represent primarily individual variation among males [12], or longlived effects of social experience. Nevertheless, this experiment indicates that social stimuli may influence levels of cortisol and prolactin in male talapoin monkeys. EXPERIMENT 2 In both the previous experiment and a recent report [34], cortisol levels of lowest-ranking male talapoin monkeys were elevated when these males received high levels of aggression. Additionally, these experiments suggest that elevated prolactin titers in higher-ranking males may be related to frequent interaction with females. Thus, social stimuli may differentially influence serum levels of cortisol and prolactin in captive talapoin monkeys. However, in both investigations, changes in socio-sexual and aggressive interactions were confounded with other variables. In Experiment 1 the housing condition changed from group-living to isolation; in the previous investigation males were tested with two different groups of females. The present study involved less drastic manipulations of the social environment. One purpose of this study was to alter the pattern of socio-sexual and aggressive interactions in a talapoin social group. The second purpose was to determine if these behavioral changes were associated with changes in males' serum levels of cortisol and prolactin. METHOD Behavioral and endocrine data were collected as described in the General Method section. This study used C-Group males for two reasons: (1) when these males were isolated, their hormone titers were rank-related (perhaps suggesting social influences), and (2) these hormone levels responded to experimental manipulation (Fig. 2). In the first

366

EBERHART, KEVERNE AND MELLER

Sot,o-sexual interactions ol males (]u rin~ selective estrogen trealment of the io;,est- rankm 9 or h=ghest- rankin 9 ~ema;e.

~lles' cortisol and prolaCtin levels Ouri n 9 selective esrtmmmmmmmtreatment mm~ ot the iowest-rarlki n 9 or r~i9nes!_ ran k~r~!en~al ~

I'0rlis01 t I

(DI

i ]

IN!

.o

Male Rank ]

4

3 ~668

T

i 604

~6

"p('02 • "p <.0~

[ ] Selectl~ estrogen to Io',~;! rtnking female (F?) en SeleCtive estr~en to highest mnldn9 female IF1)

oJ Male

FIG. 3. Socio-sexual interactions of each C-Group male during selective estrogen treatment of the lowest-ranking female (clear bars) or the highest-ranking female (shaded bars). For each treatment, data (Median_I.Q.R. per I00 minutes) represent n=16, 100-minute observations over a 4 week period. *p<0.02, **p<0.002 by Mann-Whitney U Test (2-tailed). Note scales on ordinate for different behaviors: PR=Presentations Received from females, I=Investigate females' perinea, M=Mount females.

half of the present study, C-Group males had access to the four (ovariectomized) females of their group (Table 1) when only the lowest-ranking female (F7) was estrogen-treated. In the second half of this study, males had access to these females when only the highest-ranking female (F1) was estrogen-treated. (Untreated females received no estradiolfilled silastic capsules.) The intended purpose of selective estrogen administration was to render females differentially attractive to males, thereby changing the pattern of behavioral interactions among group members. Indeed, selective estrogen treatment was associated with distinct alterations in the distribution of socio-sexual behavior directed to, and engaged in by, females [10,13]. By contrast, when all females received estrogen, the pattern of behavioral interactions between males and females of this group remained stable over 16 months of group living [10]; therefore, significant changes in behavior found in the present study may be attributed to selective estrogen treatment. Each treatment in this study represents 16, 100-minute observations, and 8 serum samples from each male, taken over a four week period. Thus, for all Mann-Whitney U tests the sample sizes are n~=n2 = 16; for all t-tests there are 14 df; and for all analyses of variance there are 3/28 df. These values are presented here to avoid repetition in the text. Both treatments were preceded by a three-four week period during which males and females had no direct contact and no observations were made; this period allowed clearance of circulating estradiol from untreated females and estradiol priming of the treated female. Consequently, in each treatment the perineal sex skin of only the treated female was swollen. RESULTS

Socio-sexual Behavior During this study (in which only one female was estrogen-treated at any time), socio-sexual interactions were less frequent than during Experiment 1 (in which all females

S

*p<,~ "'p <'001 " " p <-OO01

i t

~r[ i

"0"g

Li) N

S

J

N

El SekRlive eslroge. Io bvQsl ran kin9 lemate (flJ r l Sel~U ~1 est roqml ~ hiqttesl rankin9 ~lmlle IF IJ

FIG. 4. Mean (+S.D.) serum levels of cortisoi (at left) and protactin (at fight) for C-Group males. Time periods and data presentation as in Fig. 3; each bar represents n=8 serum samples taken over a 4 week period: *p<0.05, **p<0.001, ***p<0.0001 by unpaired t-test (2-tailed).

received estrogen). Figure 3 shows, for each male in each treatment, frequencies of presentations received from females, investigations of females' perinea, and total mounts of females. Highest-ranking S frequently participated in these interactions, and second-ranking D also engaged in each of these behaviors. Third-ranking I showed moderate levels of investigations, but lowest-ranking N rarely investigated females. Both I and N received very few female presentations, and neither of these males mounted females. No male mounted to ejaculation with any female during this study. Figure 3 also indicates that selective estrogen treatment was associated with changes in the socio-sexual behavior of two males. Highest-ranking S investigated females' sex skin more frequently, U=6.5, p<0.002, and received more presentations from females, U=I2, p<0.002, when only the highest-ranking female was estrogen-treated. By contrast, male I investigated females' sex skins more often, U=58, p<0.02, when only the lowest-ranking female received estrogen. Across treatments, there were no other statistically robust changes in males' socio-sexual interactions with females. Aggression The male dominance order was S > D > I > N , remaining stable throughout this study. Aggression among males was similar in frequency and distribution to the pattern described in Experiment 1 (Tables 2, 3): the highest-ranking male gave and received little aggression; middle-ranking males both gave and received more aggression; and the lowest-ranking male gave little, but received frequent, aggression from other males. Considering aggressive interactions between males and females, no male showed any change across treatments in the frequency of attacks, threats, or displacements given to, or received from, females, or in the frequency of withdrawals from female aggression. However, each male showed some change across treatments in the frequency of aggressive interactions with other males.

CORTISOL AND PROLACTIN IN MONKEYS When the highest-ranking female was selectively estrogen-treated, highest-ranking S both threatened other males more frequently, U=69, p<0.05, and withdrew more often from male aggression, U=69, p<0.05. In this same period, second-ranking D threatened other males more frequently, U=62, p<0.02, but attacked them less frequently, U=37, p<0.002, than during selective estrogen treatment of the lowest-ranking female. Third-ranking I displaced other males more often, U=41.5, p<0.002, and received fewer male attacks, U=51, p<0.02, when only the highest-ranking female was estrogen-treated. Finally, lowest-ranking N received more threats, U=56.5, p<0.02, and displacements, U=38.5, p<0.002, from other males at this time. Thus, there is no pattern in male-male aggression common to all males. Cortisol

The left panel of Fig. 4 presents cortisol levels of males during both treatments. When only the lowest-ranking female received estrogen, cortisol levels differed among males, F=24.8, p<0.001; cortisol levels of the two higherranking males (S, D) were similar, as were those of the two lower-ranking males (I, N). Both S and D had lower levels of cortisol at this time than did I and N (4 comparisons, Scheffr: each p<0.001). Males' cortisol titers also differed when the highest-ranking female was selectively estrogentreated, F=3.86, p =0.02. The Scheff6 procedure detects no differences among male cortisol levels at this time, but t-tests indicate that both S, t=5.092, p<0.001, and D, t=4.181, p<0.001, had significantly lower levels than N. Across treatments, each male had higher cortisol levels when the highest-ranking female was selectively estrogen-treated than when the lowest-ranking female was so treated. Males S, t=8.516, p<0.0001, and D, t=6.449, p<0.0001, showed marked elevations in cortisol; I, t=2.323, p<0.05, and N, t =4.169, p<0.001, also experienced increases. Prolactin

The right panel of Fig. 4 illustrates prolactin levels of males during both treatments. When the lowest-ranking female was selectively estrogen-treated, there were clear differences in prolactin levels among males, F=19.3, p <0.001. As with cortisol during this period, prolactin levels were similar in the two higher-ranking males, and in the two lower-ranking males. However, unlike cortisol, prolactin titers in higher-ranking males were higher than those in lowerranking males (S: 2 comparisons, p<0.001; D: 2 comparisons, p<0.005). Prolactin levels also varied among males when the highest-ranking female was selectively estrogentreated, F= 15.7, p<0.001. In this condition, second-ranking D had higher prolactin levels than any other male (3 comparisons: p <0.005). No other differences among males were statistically reliable. Considering changes in prolactin, three of four males had significantly higher prolactin titers when the lowest-ranking female was selectively estrogen-treated than when the highest-ranking female was so treated--a difference in the opposite direction to that observed with cortisol. Across treatments, highest-ranking S experienced a marked fall in prolactin levels, t=6.270, p<0.0001; both middle-ranking males showed decreases (D: t=2.529, p<0.05; I: t=2.179, p<0.05); but N exhibited no change in prolactin titers. Thus, changes in prolactin do not parallel changes in cortisol.

367 DISCUSSION The purpose of this study was to determine if selective estrogen treatment of either the highest- or lowest-ranking female would influence the social behavior or the serum levels of cortisol and prolactin in male talapoin monkeys. Although this manipulation did not change any aspect of male-female aggression, it did change, albeit inconsistently, the frequency of male-male aggression. In terms of males' participation in socio-sexual behavior, only the highestranking male showed clear changes in socio-sexual interactions; S interacted with females more frequently during selective treatment of the highest-ranking female. Considering endocrine changes, each male had higher levels of cortisol, and three of four males had lower levels of prolactin, when only the highest-ranking female was estrogen-treated. That cortisol and prolactin responses may be dissociated by a change in the social environment suggests that interpreting elevation of these hormones as a nonspecific "stress" response may be overly simplistic. Potential social influences on cortisol and prolactin levels deserve closer examination. GENERAL DISCUSSION Cortisol

A consistent pattern in the endocrine data from these studies is for the two lowest-ranking males of each group to have higher levels of cortisol when group-housed than when isolated (Fig. 2). In the social groups these males rarely interacted with females (Table 2), but received frequent aggression from other animals (Table 3). This finding is consistent with results from other endocrine studies of primates. Young rhesus monkeys showed elevations in cortisol levels that were positively correlated with the amount of aggression they received [30]; likewise, subadult rhesus monkeys receiving frequent aggression showed increased adrenal responsiveness [28,29]. It has also been suggested [6,28] that elevated adrenocortical activity may reflect, not aggression received, but "fearfulness"--i.e., an animal's response to behavioral interactions, rather than the social environment per se. In Experiment 1, among the males of Group C, which had frequent aggressive interactions during the 7 weeks of group' housing (Tables 2, 3), the highest-ranking male had the lowest cortisol levels (Figs. 1, 2). The persistence of higher cortisol levels in the other males of this group over 4 weeks of isolation may suggest that this difference reflects a long-term effect of social experience. Observations from another group of captive talapoin monkeys support this suggestion; in that report [17] cortisol titers of a lowest-ranking male increased over a year of group-living, despite a marked decrease in the frequency of aggression. Thus, the chronic receipt, or constant threat, of aggression may be sufficient to maintain elevated cortisol levels, even beyond the period of exposure to aggression. In Experiment 2, each male had higher cortisol levels when only the highest-ranking female was estrogen-treated than when the lowest-ranking female was so treated (Fig. 4). Large increases in cortisol titers were observed in the two highest-ranking males. Although all males investigated the sex skin of females (a behavior not requiring female proximity or cooperation), only the two higber-ranking males reliably received female presentations, and mounted females (Fig. 3). However, no male ejaculated with females, perhaps because females dominated males during this period [34].

368

E B E R H A R T , K E V E R N E AND M E L L E R

Despite the frequent participation by the highest-ranking female in socio-sexual interactions during estrogentreatment, this female directed aggression to males at this time (as well as during treatment of F7). Consequently, males interacting with the highest-ranking female risked receiving aggression. By contrast, when the lowest-ranking female (F7) was selectively estrogen-treated, she was not aggressive to males. Thus, increased cortisoi levels in the highest-ranking male (and perhaps in the second-ranking male) during selective estrogen-treatment of the highestranking female may be related to frequent interactions with this aggressive female. Elevated cortisol titers in the lowestranking male may reflect the more frequent aggression that he received at this time. These findings support the view that cortisol levels, while responsive to overt aggression, may also reflect the potential for such interactions. Prolactin

Males of Groups A and C i n t e r a c t e d - - a t different times---with the same females (Table 1). During isolation, the pattern of prolactin titers was similar in these groups. Prolactin levels were high in the highest-ranking male and lower in the lowest-ranking male. In these males of extreme rank, isolation levels of prolactin correspond to the frequency of socio-sexual interactions with females during group-housing (Table 2). Middle-ranking males of both groups had similar, or intermediate, levels of prolactin (Fig. 1), and of socio-sexual interactions (Table 2), particularly if investigation of females' sex skin is considered [12]. Consequently, interactions with females may be associated in some way with males' prolactin levels. As suggested for changes in cortisol levels (above), these elevated prolactin levels may outlast the period of exposure to the relevant stimuli. Although these relations between behavioral and endocrine measures are suggestive, further work in this area is clearly needed. The decrease in prolactin titers in three of four males across the treatments of Experiment 2 (Fig. 4) is not readily explained. It is possible (as above, where the potential for aggression may be associated with elevated cortisol levels)

that the potential for interaction with a receptive female may be related to elevated prolactin titers in males. The failure of males to copulate with estrogen-treated F1 was associated with this female's lack of receptivity and with female-male aggression [34]. By contrast, the lack of copulation with estrogen-treated F7 was associated with the presence of other females and the aggression they directed to this lowest-ranking female [10,13]. Unlike F I , when estrogentreated in the absence of other females, F7 was receptive and received ejaculations from these males. Thus, unlike F1, F7 was a potential partner for socio-sexual interactions. This observation is perhaps associated with the elevated prolactin titers exhibited by the three most sexually active males during selective estrogen treatment of F7 (Fig. 41. Conclusion,~

Circulating levels of corticosteroids and prolactin have been measured in primates under diverse experimental conditions. A frequent interpretation of such studies is that elevated levels of these hormones reflect a state of " s t r e s s . " In the present studies, cortisol levels were elevated in males that were frequent, or potential, recipients of aggression; in some cases, prolactin titers were elevated in males that had frequent, or potential, socio-sexuai interactions with receptive females. Under these conditions, changes in cortisol did not parallel changes in prolactin. Indeed, selective estrogen treatment of females altered cortisol and prolactin levels in opposite directions. Thus, postulating a single construct, such as " s t r e s s , " as the intervening variable mediating these endocrine changes may not always be appropriate. ACKNOWLEDGMENTS This work was supported by an MRC Program Grant to Dr. J. Herbert. We thank Dr. H. G. Friesen for prolactin antisera, Dr. R. G. Edwards for 125I-Prolactin, and the W.H.O. for prolactin standard. We also thank U. S. Yodyingyuad for invaluable assistance, G. M. Moore for statistical advice and programming, and R. Overhill for drawing the figures. J.A.E. was supported by a Marshall Scholarship and a Danforth Fellowship, and R.E.M. by an MCR Studentship.

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