Progesterone inhibition of aggressive behaviors in hamsters

Physiology & Behavior, Vol. 39, pp. 225-229. Copyright © Pergamon Journals Ltd., 1987. Printed in the U.S.A.

0031-9384/87 $3.00 + .00

Progesterone Inhibition of Aggressive Behaviors in Hamsters I. G. FRAILE, B. S. M c E W E N

A N D D. W. P F A F F

The Rockefeller University, 1230 York Avenue, N e w York, N Y 10021 R e c e i v e d 25 M a r c h 1986 FRAILE, I. G., B. S. McEWEN AND D. W. PFAFF. Progesterone inhibition of aggressive behaviors in hamsters. PHYSIOL BEHAV 39(2) 225-229, 1987.--Effects of progesterone on aggressive behaviors were tested in male-male or female-female pairs of hamsters, after both members of the pair had received the same experimental treatment. In castrated males, progesterone increased the latency to attack, and decreased the frequency of attack and on-back fighting postures. Similarly, in ovariectomized females, progesterone decreased the frequencies of upright and attack fighting responses, as well as the frequency of rolling fights. Estradiol treatment did not produce these effects on aggressive behaviors, and was not required to prime the nervous system for the progesterone effects. These results suggest that progesterone may act by means other than estrogen-inducible progestin receptors to inhibit aggressive behaviors. Progesterone

Aggression

Hamsters

G O N A D A L hormones have clear influences on social interactions between animals, especially sexual and aggressive behaviors. Among mammals and birds of many species, males are often more aggressive than females. Testicular androgens are responsible for this sex difference, and in the adult male aggressiveness increases after testosterone administration [6,10]. However, in some species, the sex difference does not exist or is reversed, as in hamsters. Among hamsters the female is more aggressive, showing high levels of agonistic behaviors outside the period of maximal sexual receptivity [7]. The literature on the effects of ovarian steroid sex hormones on the displays of aggressive behavior contain contradictions. Ovariectomy without hormone replacement has been reported to both increase [3] and decrease aggressiveness [11, 20, 21], interact with the effects of the social housing of the animal [9], or have no effect [29]. Similarly, estrogen has been reported to decrease [3], increase [11] or result in no appreciable change in aggressive behavior [20, 21, 29]. Apparently hormone dose, duration of hormone replacement, time elapsed since gonadectomy, and the nature of the behavioral tests are important methodological considerations for the nature of ovarian hormone effects on hamster aggression. A wider agreement is reached on the marked decrease in aggressiveness observed when rodent females are tested in behavioral estrous, either natural or externally induced by injecting estradiol plus progesterone in gonadectomized animals [3, 6, 7, 28, 31]. In the present work we have focused on the role played by progesterone in the modulation of the aggressive display of male and female hamsters, both in the presence (intact animals) and absence (gonadectomized animals) of an endogenous source of estrogen. The hamster model is particularly well suited for testing hypotheses about estrogen and progesterone because, if the animals are housed and tested appropriately, aggressive displays during tests are spontaneous and unreceptive

females are more aggressive than males. We have found that behavioral tests involving pairs of animals of the same sex, distinct from what has been done in some other works [1, 21, 27], facilitate the study of hormone effects on purely aggressive behaviors and their isolation from social stimuli primarily related to sexual behaviors. Pairs of animals of the same sex and belonging to the same experimental treatment groups were tested, so that all causal factors would work in the same direction. Our results show that progesterone inhibits aggression in hamsters regardless of the sex and gonadal status of the test animals. METHOD

Subjects and Maintenance The subjects in these experiments were 22 adult male and 19 female golden hamsters (Mesocricetus auratus, W., Lakeview strain from Charles River Co.) 45 days old at the time of arrival. Upon arrival animals were housed singly in Plexiglas cages in a colony room maintained on a 14 hr light, 10 hr dark cycle (lights off 12:00 noon), and at a constant temperature (about 24°C). All animals had free access to food and water.

Pairs-Composition and Estrous Cycling All animals were matched for body weight, with a maximum difference in weight of I0 g in each pair, and the composition of the pairs was kept constant throughout the study. The estrous cycle of the intact females was monitored by visual inspection and they showed a regular four-day cycle [16]. The pairs of intact females were always tested while cycling synchronously. Those pairs of animals wounded during the encounters or showing a consistent dominant/submissive pattern were excluded from statistical analyses.

225

226

FRAILE, TABLE

McEWEN

1

EFFECT OF PROGESTERONE ON THE AGGRESSIVE BEHAVIOR OF INTACT MALE AND FEMALE HAMSTERS* Latency to Upright

Latency to Attack

Upright

Attack

On-Back

RollingFight

N

24 ± 6 10 ± 35

16 ± 4 8 ± 3

9 ± 3 3 ± 2

(11) (11)

41 ± 6 37 ± 3

33 ± 4 29 ± 3

24 ± 4 19 ± 3

(9) (9)

Males Intact Intact + P

7 ± 1 49 ± 13t

299 ± 62 403 ± 67

22 ± 3 15 ± 3 Females

Intact Intact + P

15 ± 3 75 ± 24t

82 ± 21 124 ± 28

15 ± 3 13 ± 2

*The data s h o w n are the arithmetic m e a n s of the scores of the n pairs of animals ( ± S E M ) . P = P r o g e s t e r o n e administered dally (1 mg/animal) during four days. The tests were given the last two days o f treatment. t p < 0 . 0 5 ; 5p<0.01. Statistical significance o f the differences with respect to the intact group within e a c h sex, as estimated from the A N O V A described in the Method section.

TABLE

2

EFFECT OF PROGESTERONE AND ESTROGEN ON THE AGGRESSIVE BEHAVIOR OF GONADECTOMIZED MALE HAMSTERS* Latency to Upright Gdx Gdx + P G d x + EB GdxLT

17 ± 3 38 ± 13 13 ± 2 19± 4

Latency to Attack 241 491 203 205

± ± ± ±

67t 54 405 515

Upright

Attack

On-Back

12 ± 13 ± 15 ± 14±

25 ± 3 ± 26± 32 ±

17 ± 2 ± 22 ± 20±

1 1 2 2

6t 2 5t 8t

4t 1 5? 5?

RollingFight 8 ± 1 ± 10± 12 ±

3 1 3 5

N (8) (8) (8) (8)

*Average scores of the n pairs of males in each group ( ± S E M ) . G d x = A n i m a l s gonadectomized s e v e n days prior to the test. G d x + P = T h e same animals as above treated with progesterone fourteen days after g o n a d e c t o m y as described in Table 1. G d x + E B = T h e same animals as above treated with 10/xg o f estradiol benzoate daily, for four days, twenty-one days after gonadectomy. T e s t s were given the last two days of the treatment. G d x L T = T h e same animals tested again forty days after g o n a d e c t o m y . tp<0.05; 5p<0.01. Statistical significance of the differences with respect to the Gdx + P group as estimated by T u k e y ' s test of multiple c o m p a r i s o n s [15]. (For additional details see the Method section.)

TABLE

3

EFFECT OF PROGESTERONE AND ESTROGEN ON THE AGGRESSIVE BEHAVIOR OF GONADECTOMIZED FEMALE HAMSTERS*

Gdx Gdx + P G d x + EB GdxLT

Latency to Upright

Latency to Attack

Upright

Attack

On-Back

RollingFight

N

15--- 4 3 6 ± 10 12 ± 3t 14 ± 4

162 163 167 167

18 10 18 13

48 ± 28_+ 46 ± 52 ±

36 21 36 39

26 11 26 35

(8) (8) (8) (8)

± ± ± ±

32 18 37 28

± ± ± ±

It 2 3t 1

55 3 5t 65

± ± ± ±

5 3 4 5t

± ± ± ±

4t 2 3t 4§

*Average scores of the n pairs of females in each group ( ± S E M ) . Abbreviations as in Table 2. t p < 0 . 0 5 ; 5p<0.01; §p<0.001. Statistical significance of the differences with respect to the Gdx + P group as estimated by T u k e y ' s test of multiple comparisons.

AND PFAFF

P R O G E S T E R O N E AND AGGRESSION IN HAMSTERS

Testing Sequence The tests started two weeks after the animals arrived at the colony room. On the first behavioral test the animals were intact and received oil injections (0.1 ml sesame oil SC) on four consecutive days. The tests were performed in the last two days. The intact females were injected in proestrous (behavioral estrous) and tested in diestrous 1 and 2, four hours after the oil injection and three hours after the lightsoff in the colony room. The next week the animals received progesterone treatment (1 mg/0.1 ml sesame oil SC) given with the same schedule and testing sequence as above. Next, the animals were gonadectomized under Nembutal anesthesia (50 mg/kg body wt.) and allowed to recover from surgery for one week before being tested again. The gonadectomized hamsters were tested after oil and progesterone treatment as described above. After another week of rest they received a treatment of estradiol benzoate (EB, 10/zg/0.1 ml sesame oil SC)during four consecutive days, following the schedule previously used for progesterone (see above). The behavioral tests were performed in the last two days of treatment. The last hormonal treatment was given twenty-eight days after gonadectomy and a week after the previous treatment. The animals received EB injections for two days (10 tzg/0.1 ml sesame oil SC), one hour before the lights-off in the colony room and progesterone was administered on the following day (1 mg/0.1 ml sesame oil SC), also at 11:00 a.m. The test was given four hours after the last hormonal treatment. The animals were tested again more than forty days after gonadectomy, without any kind of hormonal replacement, to study the effect of time and repetition of tests on the parameters under study.

Testing Procedure The tests were conducted in the animals' own housing cages (45x20x26 cm), during two consecutive days on a " H o m e - A w a y " rotation. The scores from both days were added and considered to be the aggressive display of a given pair. Each test was five minutes in duration, in order to limit the intensity of aggression and thus prevent injury, and occurred in the colony room, three hours after lights-off, under dim red light. Testing started by placing the "intruder" in the opponent's cage, just opposite the "resident" and finished by returning the former to its home cage, and vice versa on the second day.

Behavioral Categories During the tests the overall behavior of the animals was recorded by counting and timing those postures shown in aggressive behavior sequences (see [6,7]). The parameters were divided into three categories. Pre-aggressive behavior. Upright position: the two animals were in the position, with both forepaws off the substrate showing the complete body length to its opponent, oriented one toward the other. Sometimes they exchange little pushes with their forepaws or sniffed the area around the opponent's head. Aggressive behaviors. Attack: a physical assault by one hamster upon the other. The attacker approached the partner oriented approximately perpendicular to the trunk, facing the flank, having one or both forepaws in contact with same part of the opponent's body, and sometimes biting the flank.

227 On-Back: the hamster in this posture laid on its back or side, and its opponent bent over it perpendicularly, usually sniffing or biting the flank. Rolling-Fight: this behavior represents a violent part of hamster aggressive displays. It has been described before by several authors as "fighting" or lying more or less perpendicularly. As they bit each other, the pair rolled over wildly and occasionally vocalized. Most of the time these postures occurred in the sequence listed here. Latencies. Latency to Upright: The time in seconds from the start of the test until the first upright position. If no upright positions occurred by the end of the tests, the latency was considered to be 600 sec (sum of 300 sec on each of two

tests). Latency to Attack: The time in seconds from the start of the test until the first attack position. If no attacks were scored by the end of the tests, this latency was considered to be 600 sec. These two latencies were chosen for measurement because they indicate the time the animals spent before assuming pre-aggressive or aggressive behaviors [6,7]. Lordotic postures were also scored whenever they appeared during the tests.

Statistical Analyses of the Data The data obtained under intact and gonadectomized conditions were analyzed separately, as were the data from each sex. In all cases a mixed model two-way ANOVA without replication was used, as previously described for a design in which the same individuals were tested repeatedly ([25] p. 323). The pairs of individuals were considered to be one factor, randomly representing the entire hamster population, and the hormonal treatment was taken as the second, fixed effect, factor. When the analyses showed significant hormone treatment differences, a Tukey's test [15] of multiple comparisons was used to establish which treatments were responsible for the observed differences. RESULTS The inhibitory effects of progesterone on aggression were first noted in intact hamsters of both sexes, in which it significantly increased the latencies to the Upright postures [males, F(1,10)=9.53 and females, F(1,8)=6.53, p<0.05]. The hormone also decreased the frequency of Attacks in males, F(1,10)=14.34, p<0.01. Table 1 shows a general tendency in the same direction for the other parameters under study. The tendency became statistically significant differences after the animals were gonadectomized (Tables 2 and 3), with the male group being the most affected. Gonadectomized males treated with progesterone showed longer Attack latencies, F(3,21)=6.96, p<0.01, and fewer Attacks, F(3,21)=4.74, p<0.05, and On-Back postures, F(3,21)=4.90, p<0.01. The number of Rolling-Fights also tended to decrease, although the differences were not statistically significant. In gonadectomized females (Table 3) progesterone treatment decreased the number of Upright postures, F(3,21)=4.18, p<0.05, and significantly reduced the frequencies of Attacks, F(3,21)=6.89, p<0.01, and RollingFights, F(3,21)=8.24, p<0.01. Since EB is known to induce progestin receptors [13, 17, 18] it was of interest to study its influence on the observed

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FRAILE, McEWEN AND PFAFF

effects of progesterone, but we tested first if EB had any effects of its own. EB did not influence any of the parameters under study. The animals injected with EB behaved just like they did before any hormonal treatment, i.e., more aggressively than when they were injected with progesterone (Tables 2 and 3). In contrast with its lack of effect when given alone, EB strongly potentiated the aggression-inhibitory activity of progesterone. By priming the animals with EB before progesterone treatment aggressive displays were abolished almost completely in both sexes. In males, the observed values for the relevant parameters were as follows: Attacks: 1-+1; Rolling-Fights: 1_+1; On-Back postures: 1_+1. These numbers should be compared to those in Table 2. F o r the females the data were as follows: Attacks: 0-+0; RollingFights: 0 - 0 ; On-Back postures: 0_+0. As it can be seen there was a sharp decrease from the values presented in Table 3. Lordotic behaviors were induced in both sexes accompanying the inhibition of aggression. All females and 73% of the males displayed lordotic postures during the tests. It should be noted that neither EB or progesterone alone elicited this type of sexual display. We tested for a possible "carry-over" of treatment effects by analyzing the behavior of the animals two weeks after the combined EB plus progesterone treatment. This group is referred to as Gdx LT (Long-Time after Gonadectomy) in Tables 2 and 3 and it did not behave significantly different from the Gdx group (gonadectomized, hormonally untreated animals), but there was a slight tendency towards higher aggressivity that enhanced the differences with respect to the progesterone treated group (Gdx+P) (Tables 2 and 3). DISCUSSION The results we have obtained indicate that progesterone inhibits aggression in male and female hamsters, more clearly so when the interference of endogenous gonadal hormones is removed by gonadectomy. A similar effect has been described in mice [5,12], where it was suggested that progesterone might interfere with the production of testicular androgens. This is a plausible explanation for mice but we can not apply it to the hamster model, in which females are more aggressive than males outside the sexual receptivity period [7,19], and the effects of gonadectomy are not conclusive for either sex. The literature on hamster aggressiveness is contradictory. Some authors found that gonadectomy decreased aggressivity in males [19,20], while others found it did not have any effect, in agreement with the results shown above [28,32]. Taking all the data together it does not seem that hamster aggression will fit the androgen-dependent model as defined for rats and mice. In this latter system it has been suggested recently that progesterone may increase aggression in mice by acting as an androgen agonist [30], but that would not explain the effect of progesterone shown here.

The mechanisms by which progesterone inhibits aggression in hamsters are not clear. Payne et al. [21,22] have described that in heterosexual encounters or when one of the members of the pair received progesterone injections, the female or the treated individual elicited less aggression than the opponent. The authors suggested that there could be a progesterone-dependent olfactory cue mediating the response. Erpino et al. [5] have also argued that progesterone could reduce aggressiveness in mice by inhibiting the production of aggression-promoting pheromones, and it is known that there are external glands in rodents which respond to steroids [23]. In hamsters it has been shown [8] that ultrasound emission is very important in the transition between aggressive and sexual behaviors in the female. All these data suggest that in our experimental system progesterone could act through ultrasonic or olfactory signals to inhibit the aggressive display and this possibility will be further explored. Another possible explanation would be that the reduced aggressiveness of the progesterone treated animals is related to the anesthetic properties of progestins [14,24]. We do not think this to be the case because when administered at the same doses and by the same route, alphaxalone, one of the most potent anesthetics of the progestin family [2], does not have any effect on aggression (unpublished results). This agrees with the results of Erpino et al. [4,5], who reported behavioral effects of high doses of progesterone on mice, without apparently reducing the general activity of the animals. Estrogen-induced progestin receptors [13, 17, 18] are not likely to be exclusively responsible for the observed behavioral changes following progesterone administration, even though EB greatly potentiates those effects without aggression effects of its own. The interpretation of the data is complicated by the strong sexual response elicited by the combination of the two hormones, which could be a competing response that reduced the opportunity for aggression. However the pronounced effect of progesterone on genetic males, in the absence of estradioi, clearly indicates that sexual behaviors are not the main reason behind the observed inhibition of aggressiveness, and that progesterone can act in the absence of estrogen priming. It could be that different mechanisms exist in males and females for the control of aggressive behaviors. It is possible that estrogen-induced progestin receptors may coordinate the aggressive response of females with the estrous cycle, while the non-induced population of progestin receptors in the male brain can be primarily responsible for the effects of progesterone described in this work. Recent studies suggest that progesterone can act directly on certain hypothalamic nuclei to modulate aggressive behaviors in the female [26]. We are currently assaying the levels of progestin receptors in the brains of male and female hamsters. Any correlation with measurable behavioral manifestations could help in the understanding of the behavioral effects of progesterone.

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