HORMONES
AND
Effects
12, 30-39 (1979)
BEHAVIOR
of Gonadal Steroids on Agonistic of Female Peromyscus leucopusl PHYLLIS
Department
Behavior
E. GLEASON, SANDRA D. MICHAEL, AND JOHN J. CHRISTIAN
of Biological Sciences, State University Binghamton, New York 13901
of New York,
The role of gonadal hormones in modifying agonistic behavior of female P. was examined by means of ovariectomy and treatment with estradiol benzoate (EB), progesterone (P), or testosterone propionate (TP). Aggression was lower in diestrous females than in proestrous females, and was eliminated by ovariectomy. Submissive behavior increased following ovariectomy; surgery had no effect on investigative behavior. Administration of EB had no effect on aggressive or submissive behavior, but higher dosages caused an increase in investigative and sexual behavior. Higher dosages of P increased aggression; P had no effect on submissive or investigative behavior. An increase in aggression also resulted from administration of high levels of TP. TP also caused an increase in investigative behavior, and had no effect on submissive behavior. These results may be due to direct effects of the administered hormones on behavior or to indirect effects such as a stimulation of pro&tin secretion or alteration of adrenal function.
leucopus
Hormonal effects on agonistic behavior of females of most mammalian species have been largely ignored for two basic reasons. First, females of many species are typically not aggressive; maternal aggression is generally believed to be an important exception to the general rule that females do not fight (Edwards and Rowe, 1975). Also, changes in hormonal state occurring both within and between various reproductive stages (e.g., immaturity, estrous cycling, pregnancy, and lactation), as well as interactions among the hormones have discouraged a systematic investigation of the hormonal control of agonistic behavior of females. One exception to the general rule of female nonaggressiveness can be found in golden hamsters. Work with hamsters indicates that females are aggressive toward both male and female conspecifics (Kislak and Beach, 1955; Payne and Swanson, 1970; Tiefer, 1970; Wise, 1974). Manipulation r Supported by Grant MH28286 from the National Institute of Mental Health and in part by a grant from BRSG to SUNY-Binghamton. 30 0018-506X/79/010030-10$01.00/0 Copyright All rights
Q 1979 by Academic Press. Inc. of reproduction in any form reserved.
STEROIDS
AND AGONISTIC
BEHAVIOR
31
of hormone levels in female hamsters through ovariectomy and administration of exogenous hormones has produced varying results, but it appears that one or more of the gonadal steroids can modify aggressive behavior (Grelk, Papson, Cole, and Rowe, 1974; Kislak and Beach, 1955; Payne and Swanson, 1971a,b, 1972; Tiefer, 1970; Vandenbergh, 1971). Adult female Peromyscus feucopus have frequently been reported to exhibit aggressive behavior toward both mature and juvenile male and female conspecifics (Burt, 1940; Nicholson, 1941; Sheppe, 1966a,b; Snyder, 1956), as well as toward both males and females of other species (Murie, 1971; Rowley and Christian, 1976). Field work has also indicated that P. feucopus of the same sex tend to occupy mutually exclusive home ranges, with resident females less tolerant of female intruders than resident males are of male intruders (Metzgar, 1971; Sheppe, 1966b). The purpose of this study is to examine the role played by gonadal steroids in altering agonistic behavior of adult female P. feucopus. Effects of estrogen, progesterone, and testosterone on aggressive, submissive, and investigative behavior of female P. leucopus interacting with female conspecifics were studied as an initial step in elucidating the role of hormones in modifying agonistic behavior in this species. METHODS Animals. Animals used in this experiment were 280 nulliparous female P. leucopus, 70-90 days of age, descended from populations trapped in
Franklin County, Pennsylvania, in 1965-1968 and subsequently maintained in the laboratory using random outbreeding techniques. Animals were weaned at 21 days and reared with one same-sex sibling until treatment. All animals were isolated at least 10, but not more than 13 days, prior to behavioral testing. Animals were maintained in 20 x 25-cm stainless-steel cages, with food and water supplied ad lib. The light cycle was white light 0500 to 1900 hr and dim red light 1900 to 0500 hr. Behavioral observations. All observations were conducted during the first 3 hr of the dark phase of the light cycle. The observation arena was a 25 x 50-cm glass aquarium with opaque shields on three sides and top and with a removable partition in the center. The approximately 3 cm wood shavings were changed and the arena was washed following each observation. At the initiation of each observation, experimental, and opponent animals were placed in the two compartments of the arena and allowed 15 min for habituation. After this, the partition was removed and the animals were allowed to interact for 15 min. During this time, the frequency and duration of each behavioral pattern listed below which was initiated by the experimental animal was recorded, using an event recorder and manual keyboard. The following behaviors were recorded: (1) facial investiga-
32
GLEASON,
MICHAEL,
AND
CHRISTIAN
tion; (2) ano-genital investigation; (3) lunge: (4) attack; (5) fight; (6) chase; (7) flee; (8) crouch. Experimental design and procedure. Forty animals were randomly assigned to four control groups: intact diestrus; intact proestrus; ovariectomized (ovx); or ovx plus oil injection (ovx-oil). One hundred animals were also randomly assigned to 10 experimental groups which were ovariectomized and then injected with either estradiol benzoate (EB), progesterone (P), or testosterone propionate (TP) at the following dosages: 0.1, 1.O, 5.0, or 20.0 pg EB; 100,250, or 500 pg P; 10, 100, or 200 pg TP. The remaining 140 animals were randomly assigned to be used as intact opponent animals in the tests. All opponent animals were used only while in diestrus, since pilot studies indicated that diestrus females exhibited relatively low levels of aggression and could elicit aggression from any intact animal with which they were tested. In no case was an animal used more than once, either as an opponent or a test animal. Vaginal smears were taken daily from all intact control and opponent animals by saline lavage and examined in unstained wet preparations. Estrous cycles were followed through at least two complete 4- or 5-day vaginal cycles and until they reached the appropriate stage in the third cycle. Smears were also taken from the intact animals immediately after behavioral testing to insure that the animal was in the appropriate stage. Bilateral ovariectomies were performed under ether anesthesia. Animals were then isolated until observations on the 10th day postsurgery. Animals in the experimental groups were injected subcutaneously with either 0.1 ml sesame oil or the designated dosage of steroid suspended in 0.1 ml sesame oil approximately 12 hr prior to observation. Following observations, all ovariectomized animals were sacrificed and examined to guarantee that surgery had been complete. Treatment of data. Behavioral patterns recorded were grouped into three different types of behavior: aggressive, submissive, and socialinvestigative. Aggressive behavior included lunges, attacks, fights, and chases. Submissive behavior included fleeing and crouching, and socialinvestigative behavior included facial and ano-genital investigation. Two measurements were recorded for each behavior type. Frequency indicated the number of behavioral acts of each type observed during the observation period, and percentage of time indicated the percentage of the total time (15 min) in which the behavioral act was occurring. A dominance score was also established for each experimental animal. This score was calculated as the number of encounters won by the experimental animal (opponent responded submissively) minus the number of encounters lost by the experimental animal (experimental animal responded submissively). One-way analyses of variance were performed on all sets of data from
33
STEROIDS AND AGONISTIC BEHAVIOR
the four control groups. If a significant difference among the four groups was indicated, the analyses were extended by a Sheffe test. Each of the experimental groups was compared with each of the control groups by a series of selected contrasts using weighted means. Since the analyses of variance, followed by Sheffe tests, indicated no difference between ovx and ovx-oil animals for any of the behavioral parameters, these groups were pooled for the selected comparisons. RESULTS
Figure 1 shows how gonadal steroids affected aggressive behavior of female P. Icucopus. The four control groups differed both in the frequency [F (3,36) = 54.52, p < .OOl] and percentage of time [F (3,36) = 35.63, p < .OOl] of aggressive acts. Proestrous females spent more time in
11 10 9 :
0
t= Y
0
7 6 5 4 3 2 1
DI
PROOVX
OIL
El
E2
E3
TREATMENT
E4
Pl
P2
P3
Tl
T2
T3
GROUP
FIG. I. Frequency and percentage of time of aggressive acts. PRO = proestrus, DI = diestrus, OVX = ovariectomized, OIL = oil-injected ovx; El, E2, E3, and E4 are ovx animals injected with 0.5, 1.0, 5.0, and 20.0 pg EB, respectively; PI, P2, and P3 are ovx animals injected with 100, 250, and 500 pg P; and Tl, T2, and T3 are animals injected with 10, 100, and 200 pg TP.
34
GLEASON,
MICHAEL,
AND CHRISTIAN
aggressive behavior than diestrous females (p < .05) but their frequency of aggression was not greater (p > .05). Ovx and ovx-oil females were less aggressive than intact animals (p < .05) by either measure, but did not differ from each other.@ > .05). EB-Treated animals were no more aggressive than ovx-oil animals by either measure [F (1,126) < 2, p > .05, for all comparisons], nor were animals injected with 100 Fg P [F (1,126) < 1, p > .05 for both measures]. Animals injected with 250 pg P were more aggressive than ovx controls [F (1,126) = 43.7, p < .OOl for frequency, F (1,126) = 6.44, p < .05 for percentage of time]; these animals were as aggressive as diestrous controls, as indicated by both frequency of aggressive acts [F (I ,126) = 1.21, p > .053and percent of time of aggressive acts [F (1,126) = 1.23, p > .051. Treatment with 500 pg P also caused marked increases in aggression, in comparison with ovx controls [F (1,126) > 200, p < .OOl for both measures]. Animals in this treatment group were more aggressive than proestrouscontrols[F(1,126) = 141.81,~ < .OOl,forfrequency;F(l,l26) = 46.29, p < .OOl for percentage of time]. Animals treated with 10 or 100 pg TP were no more aggressive than ovx controls [F (1,126) < 2.16, p > .05 for all comparisons]. Injection of 200 pg TP, however, caused an increase in the frequency of aggression [F (1,126) = 33.84, p < .OOl]; in this group both frequency and percentage of time of aggressive acts were similar to diestrous controls [F (1,126) = 1.31, p > .05, and F (1,126) = 1.12, p > .05, respectively]. Submissive behavior was also affected by hormonal manipulations (Fig. 2). Ovariectomy caused an increase in the frequency of submissive acts [F (3,36) = 2.86, .lO < p > .05] and in the percentage of time of submissive behavior [F (3,36) = 7.54, p < .OOl] when compared to intact control groups. There were no differences between the two ovx control groups or the two intact control groups (p > .05 in all cases) indicated by either measure of submissiveness. Only animals injected with 250 pg P were less submissive than ovx controls [F (1,126) = 5.82, p < .05 for frequency, F (1,126) = 9.33,~ < .OOl for percentage of time]. Animals treated with EB were as submissive as ovx controls [F (1,126) < 2.37, p > .05 for all comparisons]. P and TP caused slight decreases in both frequency and percentage of time of submissive behavior, in comparison with ovx controls. Both measures were reduced to levels intermediate between ovx controls and intact controls and were not different from either control group [F (1,126) < 2.48, p > .05, for all comparisons]. Effects of hormone treatments on investigative behavior are shown in Fig. 3. Neither frequency nor percentage of time of investigative behavior was different among the control groups [F (3,36) = 2.53, p > .05, and F (3,36) = 2.66, p > .05, respectively]. A significant increase in both measures of investigative behavior resulted from the administration of the
STEROIDS
AND AGONISTIC
TREATMENT
35
BEHAVIOR
GROUP
FIG. 2. Frequency and percentage of time of submissive acts. Treatment groups are indicated as in Fig. 1.
three higher dosages of EB and from the administration of TP [F (1,126) < 3.99, p < .05 for all comparisons]. This increase was accompanied by an increase in sexual behavior. Dominance relationships between experimental and opponent animals were also affected by the various hormonal treatments (Table 1). Dominance scores of ovx control animals were lower than those of intact
DI PROOVXOIL
El
E2
E3
TREATMENT
E4
Pl
P2
P3
Tl
T2
T3
GROUP
FIG. 3. Frequency and percentage of time of investigative acts. Treatment groups are indicated as in Fig. 1.
36
GLEASON, MICHAEL,
AND CHRISTIAN
TABLE 1 Effects of Gonadal Steroids on Dominance Scores: Dominance Scores of Intact and Hormone-Treated Females Experimental
group
Dominance score (?SEM)
Diestrus Proestrus ovx Ovx-oil EB 0.5 PcLg 1.0 /G 5.0 M 20.0 /.Lg P 100 Pk? 250 pg 500 /a TP 19 I% 100 fig 250 ia
0.9 4.0 -5.8 -4.8
t + zt +
0.6 1.2 1.9 1.6
-6.2 -4.7 -4.0 -4.8
!c + 2 r
1.7 1.5 1.5 1.1
-4.2 2 2.1 2.5 r 1.1 7.5 5 2.2 -4.2 k 1.1 -3.4 2 1.1 -0.2 t 1.8
control animals [F (3,36) = 34.39, p < .OOl]; proestrous animals had higher dominance scores than diestrous animals (p < .05). Animals injected with EB at any dosage, 100 pg P or 10 or 100 pg TP did not have dominance scores higher than those of ovx animals [F (1,126) = 1.65, p > .05]. Dominance scores of animals treated with 250 pg P or 200 pg TP were comparable to those of diestrous animals [F (1,126) = 1.88, p > .05, for P; F (1,126) = 1.42, p > .05 for TP], and dominance scores of animals injected with 500 pg P were higher than those of proestrous animals [F (1,126) = 14.52, p < .OOl]. DISCUSSION
The results of these experiments indicate that estradiol, progesterone, and testosterone can differentially modify aspects of agonistic behavior of female P. leucopus. Removal of the steroids through ovariectomy caused an increase in submissive behavior; this effect was partially overcome by administration of progesterone or testosterone. Since no hormone combinations were tested, the possibility that a synergistic action between two or more hormones might reverse the effects of ovariectomy on submissive behavior cannot be ignored. It is also possible, however, that aggressive and submissive behavior are influenced by different hormones and mechanisms, as suggested by Leshner and Moyer (1975) and Leshner, Moyer, and Walker (1975) for male house mice. The greatest effects of the hormone treatments on agonistic behavior
STEROIDS
AND AGONISTIC
BEHAVIOR
37
were observed in the measures of aggression and dominance. Ovariectomy eliminated aggressive behavior; levels of aggression comparable to or higher than those observed in intact females resulted from administration of progesterone or high dosages of testosterone. There are some similarities between these results and those obtained in similar experiments working with female golden hamsters. For example, Grelk rr al. (1974), Payne and Swanson (1971b, 1972), and Tiefer (1970) found that estrogen had no effect on aggressive behavior, and Payne and Swanson (1971a) reported a decrease in aggressive behavior resulting from injection of high dosages of estrogen. Testosterone administered to female hamsters has typically caused no change in aggressive behavior (Payne and Swanson 1971a, b, 1972; Tiefer, 1970; Vandenbergh, 1971), although Grelk et al. (1974) found that testosterone caused increases in aggressive behavior in group-housed ovariectomized animals. Progesterone also can stimulate aggression (Payne and Swanson, 1971a, b, 1972) although Grelk et al. (1974) and Kislak and Beach (1955) found that progesterone had no effect. Many of the inconsistencies found in work on the hormonal control of behavior in females are undoubtedly due to methodological differences. However, because of the extremely complex, cyclic nature of the female endocrine system, an effect of a single exogenous hormone on behavior cannot be taken as proof that that particular hormone regulates the behavior in normal intact animals. First, as mentioned previously, synergistic actions among hormones are undoubtedly important. Second, the observed results may be indirect effects mediated by the secretion of some other hormone. For example, Christian’s (1971) report that adrenal weight in both male and female P. leucopus was above average during the breeding season suggests a possible influence of the reproductive hormones on adrenal function. Altered adrenal function, in turn, may be an important factor in the modification of agonistic behavior (cf. Edwards and Rowe, 1975). Also, Chen and Meites (1970) and Kalra, Fawcett, Krulich, and McCann (1973) found that estrogen, progesterone, and testosterone stimulated prolactin secretion in rats. Therefore, the effects we observed might also be due to stimulated prolactin secretion. Wise (1974) examined aggression during various reproductive stages of female hamsters and found that changes in aggression corresponded to changes in pituitary prolactin levels (Kent, 1968), including times when the ovaries are quiescent (Greenwald, 1965)and facilitation of aggression by ovarian hormones is unlikely. Noirot, Goyens, and Buhot (1975) reported patterns of aggression in pregnant and pseudopregnant female mice which correspond to increasing progesterone (Murr, Stabenfeldt, Bradford, and Geschwind, 1974) and prolactin (Murr, Bradford, and Geschwind, 1974) levels. Lactat-
38
GLEASON, MICHAEL,
AND CHRISTIAN
ing mice also exhibit patterns of aggression (Gandelman, 1972; St. John and Corning, 1973; Svare and Gandelman, 1973) which are similar to changes in circulating levels of prolactin in lactating mice (Sinha, Selby, and VanderLaan, 1974). This experiment has shown that gonadal steroids can affect the agonistic behavior of female P. leucopus. Further investigation is clearly necessary, however, to clarify the role of other hormones and hormone interactions in behavioral regulation in this species. REFERENCES Burt, W. H. (1940). Territorial behavior and populations of some small mammals in southern Michigan. Misc. Pub/. Mus. Zoo/. Univ. Mich. 45, 58~. Chen, C. L., and Meites, J. (1970). Effects of estrogen and progesterone on serum and pituitary prolactin levels in ovariectomized rats. Endocrinology 86, 503-505. Christian, J. J. (1971). Population density and reproductive efficiency. Biol. Reprod. 4,248. Edwards, D. A., and Rowe, F. A. (1975). Neural and endocrine control of aggressive behavior. In B. E. Eleftheriou and R. L. Sprott (Eds.), Hormonal Correlates of Behavior, Vol. 1. Plenum, New York. Gandelman, R. (1972). Mice: Postpartum aggression elicited by the presence of an intruder. Horm.
Behav. 3, 23-28.
Greenwald, G. S. (1%5). Histologic transformation of the ovary of the lactating hamster. Endocrinology
77, 641-650.
Grelk, D. F., Papson, B. A., Cole, J. E., and Rowe, F. A. (1974). The influence of caging conditions and hormone treatments on fighting in male and female hamsters. Horm. Behav.
5, 355-366.
Kalra, P. S., Fawcett, C. P., Krulich, L., and McCann, S. M. (1973). The effects of gonadal steroids on plasma gonadotropins and prolactin in the rat. Endocrino/ogy 92, 12561268. Kent, G. C. (1968). Physiology and reproduction. In R. A. Hoffman, P. F. Robinson, and H. Magalhaes (Eds.), The Golden Hamster: Its Biology and Use in Medical Research. Iowa Univ. Press, Ames. Kislak, J. W., and Beach, F. A. (1955). Inhibition of aggressiveness by ovarian hormones. Endocrinology
56, 684-692.
Leshner, A. I., and Moyer, J. A. (1975). Androgens and agonistic behavior of mice: Relevance to aggression and irrelevance to avoidance-of-attack. Physiol. Behav. 15, 695-699. Leshner, A. I., Moyer, J. A., and Walker, W. A. (1975). Pituitary-adrenocortical activity and avoidance-of-attack in mice. Physiol Behav. 15, 689-693. Metzgar, L. H. (1971). Behavioral population regulation in the woodmouse, Peromyscus leucopus.
Amer. Midl.
Natur.
86, 434-448.
Murie, J. 0. (1971). Dominance relationships between Peromyscus and Microtus in captivity. Amer. Midl. Natur. 86, 229-230. Murr, S. M., Bradford, G. E., and Geschwind, I. I. (1974). Plasma luteinizing hormone, follicle stimulating hormone, and prolactin during pregnancy in the mouse. Endocrinology
94, 112-I 16.
Murr, S. M., Stabenfeldt, G. H., Bradford, G. E., and Geschwind, I. 1. (1974). Plasma progesterone during pregnancy in the mouse. Endocrinology 94, 1209-1211. Nicholson, A. J. (1941). The homes and social habits of the wood mouse (P. leucopus noveboracensis) in southern Michigan. Amer. Midl. Natur. 25, 196-223.
STEROIDS AND AGONISTIC BEHAVIOR
39
Noirot, E., Goyens, J., and Buhot, M. C. (197.5). Aggressive behavior of pregnant mice toward males. Horm. Behav. 6, 9-17. Payne, A. P., and Swanson, H. H. (1970). Agonistic behaviour between pairs of hamsters of the same and opposite sex in a neutral observation arena. Behaviour 36, 259-269. Payne, A. P., and Swanson, H. H. (1971a). Hormonal control of aggressive dominance in the female hamster. Physiol. Behav. 6, 355-357. Payne, A. P., and Swanson, H. H. (1971b). Hormonal modification of aggressive behavior between female golden hamsters. J. Endocrinol. 51, xvii-xviii. Payne, A. P., and Swanson, H. H., (1972). The effect of sex hormones on the aggressive behavior of the female golden hamster (Mesocricetus auratus Waterhouse). Anim. Behav.
20, 782-787.
Rowley, M. H., and Christian, J. J. (1976). Interspecific aggression between Peromyscus and Microrus females: A possible factor in competitive exclusion. Behav. Biol. 16, 521-525.
St. John, R. D., and Corning, P. A. (1973). Maternal aggression in mice. Behav. Biol. 9, 635-639.
Sheppe, W. A. (1%6a). Social behavior of the deermouse Peromyscus leucopus in the laboratory. Wasmann J. Biol. 24, 49-65. Sheppe, W. A. (1%6b). Determinants of homerange in the deer mouse, Peromyscus Ieucopus.
Proc. Calif. Acad. Sci. 34, 377-418.
Sinha, Y. N., Selby, F. W., and VanderLaan, W. P. (1974). Relationship of prolactin and growth hormone to mammary function during pregnancy and lactation in the C3H/ST mouse. J. Endocrinol. 61, 219-229. Snyder, D. P. (1956). Survival rates, longevity, and population fluctuations in the whitefooted mouse, P. leucopus, in southeastern Michigan. Misc. Publ. Mus. Zool. Univ. Mich. 95, 33~. Svare, B., and Gandelman, R. (1973). Postpartum aggression in mice: Experiential and environmental factors. Horm. Behav. 4, 323-334. Tiefer, L. (1970). Gonadal hormones and mating behavior in the golden hamster. Horm. Behav.
1, 189-202.
Vandenbergh, J. G. (1971). The effects of gonadal hormones on the aggressive behaviour of adult golden hamsters (Mesocricetus auratus). Anim. Behav. 19, 589-594. Wise, D. A. (1974). Aggression in the female golden hamster: Effects of reproductive state and social isolation. Horm. Behav. 5, 235-250.
AND
Effects
12, 30-39 (1979)
BEHAVIOR
of Gonadal Steroids on Agonistic of Female Peromyscus leucopusl PHYLLIS
Department
Behavior
E. GLEASON, SANDRA D. MICHAEL, AND JOHN J. CHRISTIAN
of Biological Sciences, State University Binghamton, New York 13901
of New York,
The role of gonadal hormones in modifying agonistic behavior of female P. was examined by means of ovariectomy and treatment with estradiol benzoate (EB), progesterone (P), or testosterone propionate (TP). Aggression was lower in diestrous females than in proestrous females, and was eliminated by ovariectomy. Submissive behavior increased following ovariectomy; surgery had no effect on investigative behavior. Administration of EB had no effect on aggressive or submissive behavior, but higher dosages caused an increase in investigative and sexual behavior. Higher dosages of P increased aggression; P had no effect on submissive or investigative behavior. An increase in aggression also resulted from administration of high levels of TP. TP also caused an increase in investigative behavior, and had no effect on submissive behavior. These results may be due to direct effects of the administered hormones on behavior or to indirect effects such as a stimulation of pro&tin secretion or alteration of adrenal function.
leucopus
Hormonal effects on agonistic behavior of females of most mammalian species have been largely ignored for two basic reasons. First, females of many species are typically not aggressive; maternal aggression is generally believed to be an important exception to the general rule that females do not fight (Edwards and Rowe, 1975). Also, changes in hormonal state occurring both within and between various reproductive stages (e.g., immaturity, estrous cycling, pregnancy, and lactation), as well as interactions among the hormones have discouraged a systematic investigation of the hormonal control of agonistic behavior of females. One exception to the general rule of female nonaggressiveness can be found in golden hamsters. Work with hamsters indicates that females are aggressive toward both male and female conspecifics (Kislak and Beach, 1955; Payne and Swanson, 1970; Tiefer, 1970; Wise, 1974). Manipulation r Supported by Grant MH28286 from the National Institute of Mental Health and in part by a grant from BRSG to SUNY-Binghamton. 30 0018-506X/79/010030-10$01.00/0 Copyright All rights
Q 1979 by Academic Press. Inc. of reproduction in any form reserved.
STEROIDS
AND AGONISTIC
BEHAVIOR
31
of hormone levels in female hamsters through ovariectomy and administration of exogenous hormones has produced varying results, but it appears that one or more of the gonadal steroids can modify aggressive behavior (Grelk, Papson, Cole, and Rowe, 1974; Kislak and Beach, 1955; Payne and Swanson, 1971a,b, 1972; Tiefer, 1970; Vandenbergh, 1971). Adult female Peromyscus feucopus have frequently been reported to exhibit aggressive behavior toward both mature and juvenile male and female conspecifics (Burt, 1940; Nicholson, 1941; Sheppe, 1966a,b; Snyder, 1956), as well as toward both males and females of other species (Murie, 1971; Rowley and Christian, 1976). Field work has also indicated that P. feucopus of the same sex tend to occupy mutually exclusive home ranges, with resident females less tolerant of female intruders than resident males are of male intruders (Metzgar, 1971; Sheppe, 1966b). The purpose of this study is to examine the role played by gonadal steroids in altering agonistic behavior of adult female P. feucopus. Effects of estrogen, progesterone, and testosterone on aggressive, submissive, and investigative behavior of female P. leucopus interacting with female conspecifics were studied as an initial step in elucidating the role of hormones in modifying agonistic behavior in this species. METHODS Animals. Animals used in this experiment were 280 nulliparous female P. leucopus, 70-90 days of age, descended from populations trapped in
Franklin County, Pennsylvania, in 1965-1968 and subsequently maintained in the laboratory using random outbreeding techniques. Animals were weaned at 21 days and reared with one same-sex sibling until treatment. All animals were isolated at least 10, but not more than 13 days, prior to behavioral testing. Animals were maintained in 20 x 25-cm stainless-steel cages, with food and water supplied ad lib. The light cycle was white light 0500 to 1900 hr and dim red light 1900 to 0500 hr. Behavioral observations. All observations were conducted during the first 3 hr of the dark phase of the light cycle. The observation arena was a 25 x 50-cm glass aquarium with opaque shields on three sides and top and with a removable partition in the center. The approximately 3 cm wood shavings were changed and the arena was washed following each observation. At the initiation of each observation, experimental, and opponent animals were placed in the two compartments of the arena and allowed 15 min for habituation. After this, the partition was removed and the animals were allowed to interact for 15 min. During this time, the frequency and duration of each behavioral pattern listed below which was initiated by the experimental animal was recorded, using an event recorder and manual keyboard. The following behaviors were recorded: (1) facial investiga-
32
GLEASON,
MICHAEL,
AND
CHRISTIAN
tion; (2) ano-genital investigation; (3) lunge: (4) attack; (5) fight; (6) chase; (7) flee; (8) crouch. Experimental design and procedure. Forty animals were randomly assigned to four control groups: intact diestrus; intact proestrus; ovariectomized (ovx); or ovx plus oil injection (ovx-oil). One hundred animals were also randomly assigned to 10 experimental groups which were ovariectomized and then injected with either estradiol benzoate (EB), progesterone (P), or testosterone propionate (TP) at the following dosages: 0.1, 1.O, 5.0, or 20.0 pg EB; 100,250, or 500 pg P; 10, 100, or 200 pg TP. The remaining 140 animals were randomly assigned to be used as intact opponent animals in the tests. All opponent animals were used only while in diestrus, since pilot studies indicated that diestrus females exhibited relatively low levels of aggression and could elicit aggression from any intact animal with which they were tested. In no case was an animal used more than once, either as an opponent or a test animal. Vaginal smears were taken daily from all intact control and opponent animals by saline lavage and examined in unstained wet preparations. Estrous cycles were followed through at least two complete 4- or 5-day vaginal cycles and until they reached the appropriate stage in the third cycle. Smears were also taken from the intact animals immediately after behavioral testing to insure that the animal was in the appropriate stage. Bilateral ovariectomies were performed under ether anesthesia. Animals were then isolated until observations on the 10th day postsurgery. Animals in the experimental groups were injected subcutaneously with either 0.1 ml sesame oil or the designated dosage of steroid suspended in 0.1 ml sesame oil approximately 12 hr prior to observation. Following observations, all ovariectomized animals were sacrificed and examined to guarantee that surgery had been complete. Treatment of data. Behavioral patterns recorded were grouped into three different types of behavior: aggressive, submissive, and socialinvestigative. Aggressive behavior included lunges, attacks, fights, and chases. Submissive behavior included fleeing and crouching, and socialinvestigative behavior included facial and ano-genital investigation. Two measurements were recorded for each behavior type. Frequency indicated the number of behavioral acts of each type observed during the observation period, and percentage of time indicated the percentage of the total time (15 min) in which the behavioral act was occurring. A dominance score was also established for each experimental animal. This score was calculated as the number of encounters won by the experimental animal (opponent responded submissively) minus the number of encounters lost by the experimental animal (experimental animal responded submissively). One-way analyses of variance were performed on all sets of data from
33
STEROIDS AND AGONISTIC BEHAVIOR
the four control groups. If a significant difference among the four groups was indicated, the analyses were extended by a Sheffe test. Each of the experimental groups was compared with each of the control groups by a series of selected contrasts using weighted means. Since the analyses of variance, followed by Sheffe tests, indicated no difference between ovx and ovx-oil animals for any of the behavioral parameters, these groups were pooled for the selected comparisons. RESULTS
Figure 1 shows how gonadal steroids affected aggressive behavior of female P. Icucopus. The four control groups differed both in the frequency [F (3,36) = 54.52, p < .OOl] and percentage of time [F (3,36) = 35.63, p < .OOl] of aggressive acts. Proestrous females spent more time in
11 10 9 :
0
t= Y
0
7 6 5 4 3 2 1
DI
PROOVX
OIL
El
E2
E3
TREATMENT
E4
Pl
P2
P3
Tl
T2
T3
GROUP
FIG. I. Frequency and percentage of time of aggressive acts. PRO = proestrus, DI = diestrus, OVX = ovariectomized, OIL = oil-injected ovx; El, E2, E3, and E4 are ovx animals injected with 0.5, 1.0, 5.0, and 20.0 pg EB, respectively; PI, P2, and P3 are ovx animals injected with 100, 250, and 500 pg P; and Tl, T2, and T3 are animals injected with 10, 100, and 200 pg TP.
34
GLEASON,
MICHAEL,
AND CHRISTIAN
aggressive behavior than diestrous females (p < .05) but their frequency of aggression was not greater (p > .05). Ovx and ovx-oil females were less aggressive than intact animals (p < .05) by either measure, but did not differ from each other.@ > .05). EB-Treated animals were no more aggressive than ovx-oil animals by either measure [F (1,126) < 2, p > .05, for all comparisons], nor were animals injected with 100 Fg P [F (1,126) < 1, p > .05 for both measures]. Animals injected with 250 pg P were more aggressive than ovx controls [F (1,126) = 43.7, p < .OOl for frequency, F (1,126) = 6.44, p < .05 for percentage of time]; these animals were as aggressive as diestrous controls, as indicated by both frequency of aggressive acts [F (I ,126) = 1.21, p > .053and percent of time of aggressive acts [F (1,126) = 1.23, p > .051. Treatment with 500 pg P also caused marked increases in aggression, in comparison with ovx controls [F (1,126) > 200, p < .OOl for both measures]. Animals in this treatment group were more aggressive than proestrouscontrols[F(1,126) = 141.81,~ < .OOl,forfrequency;F(l,l26) = 46.29, p < .OOl for percentage of time]. Animals treated with 10 or 100 pg TP were no more aggressive than ovx controls [F (1,126) < 2.16, p > .05 for all comparisons]. Injection of 200 pg TP, however, caused an increase in the frequency of aggression [F (1,126) = 33.84, p < .OOl]; in this group both frequency and percentage of time of aggressive acts were similar to diestrous controls [F (1,126) = 1.31, p > .05, and F (1,126) = 1.12, p > .05, respectively]. Submissive behavior was also affected by hormonal manipulations (Fig. 2). Ovariectomy caused an increase in the frequency of submissive acts [F (3,36) = 2.86, .lO < p > .05] and in the percentage of time of submissive behavior [F (3,36) = 7.54, p < .OOl] when compared to intact control groups. There were no differences between the two ovx control groups or the two intact control groups (p > .05 in all cases) indicated by either measure of submissiveness. Only animals injected with 250 pg P were less submissive than ovx controls [F (1,126) = 5.82, p < .05 for frequency, F (1,126) = 9.33,~ < .OOl for percentage of time]. Animals treated with EB were as submissive as ovx controls [F (1,126) < 2.37, p > .05 for all comparisons]. P and TP caused slight decreases in both frequency and percentage of time of submissive behavior, in comparison with ovx controls. Both measures were reduced to levels intermediate between ovx controls and intact controls and were not different from either control group [F (1,126) < 2.48, p > .05, for all comparisons]. Effects of hormone treatments on investigative behavior are shown in Fig. 3. Neither frequency nor percentage of time of investigative behavior was different among the control groups [F (3,36) = 2.53, p > .05, and F (3,36) = 2.66, p > .05, respectively]. A significant increase in both measures of investigative behavior resulted from the administration of the
STEROIDS
AND AGONISTIC
TREATMENT
35
BEHAVIOR
GROUP
FIG. 2. Frequency and percentage of time of submissive acts. Treatment groups are indicated as in Fig. 1.
three higher dosages of EB and from the administration of TP [F (1,126) < 3.99, p < .05 for all comparisons]. This increase was accompanied by an increase in sexual behavior. Dominance relationships between experimental and opponent animals were also affected by the various hormonal treatments (Table 1). Dominance scores of ovx control animals were lower than those of intact
DI PROOVXOIL
El
E2
E3
TREATMENT
E4
Pl
P2
P3
Tl
T2
T3
GROUP
FIG. 3. Frequency and percentage of time of investigative acts. Treatment groups are indicated as in Fig. 1.
36
GLEASON, MICHAEL,
AND CHRISTIAN
TABLE 1 Effects of Gonadal Steroids on Dominance Scores: Dominance Scores of Intact and Hormone-Treated Females Experimental
group
Dominance score (?SEM)
Diestrus Proestrus ovx Ovx-oil EB 0.5 PcLg 1.0 /G 5.0 M 20.0 /.Lg P 100 Pk? 250 pg 500 /a TP 19 I% 100 fig 250 ia
0.9 4.0 -5.8 -4.8
t + zt +
0.6 1.2 1.9 1.6
-6.2 -4.7 -4.0 -4.8
!c + 2 r
1.7 1.5 1.5 1.1
-4.2 2 2.1 2.5 r 1.1 7.5 5 2.2 -4.2 k 1.1 -3.4 2 1.1 -0.2 t 1.8
control animals [F (3,36) = 34.39, p < .OOl]; proestrous animals had higher dominance scores than diestrous animals (p < .05). Animals injected with EB at any dosage, 100 pg P or 10 or 100 pg TP did not have dominance scores higher than those of ovx animals [F (1,126) = 1.65, p > .05]. Dominance scores of animals treated with 250 pg P or 200 pg TP were comparable to those of diestrous animals [F (1,126) = 1.88, p > .05, for P; F (1,126) = 1.42, p > .05 for TP], and dominance scores of animals injected with 500 pg P were higher than those of proestrous animals [F (1,126) = 14.52, p < .OOl]. DISCUSSION
The results of these experiments indicate that estradiol, progesterone, and testosterone can differentially modify aspects of agonistic behavior of female P. leucopus. Removal of the steroids through ovariectomy caused an increase in submissive behavior; this effect was partially overcome by administration of progesterone or testosterone. Since no hormone combinations were tested, the possibility that a synergistic action between two or more hormones might reverse the effects of ovariectomy on submissive behavior cannot be ignored. It is also possible, however, that aggressive and submissive behavior are influenced by different hormones and mechanisms, as suggested by Leshner and Moyer (1975) and Leshner, Moyer, and Walker (1975) for male house mice. The greatest effects of the hormone treatments on agonistic behavior
STEROIDS
AND AGONISTIC
BEHAVIOR
37
were observed in the measures of aggression and dominance. Ovariectomy eliminated aggressive behavior; levels of aggression comparable to or higher than those observed in intact females resulted from administration of progesterone or high dosages of testosterone. There are some similarities between these results and those obtained in similar experiments working with female golden hamsters. For example, Grelk rr al. (1974), Payne and Swanson (1971b, 1972), and Tiefer (1970) found that estrogen had no effect on aggressive behavior, and Payne and Swanson (1971a) reported a decrease in aggressive behavior resulting from injection of high dosages of estrogen. Testosterone administered to female hamsters has typically caused no change in aggressive behavior (Payne and Swanson 1971a, b, 1972; Tiefer, 1970; Vandenbergh, 1971), although Grelk et al. (1974) found that testosterone caused increases in aggressive behavior in group-housed ovariectomized animals. Progesterone also can stimulate aggression (Payne and Swanson, 1971a, b, 1972) although Grelk et al. (1974) and Kislak and Beach (1955) found that progesterone had no effect. Many of the inconsistencies found in work on the hormonal control of behavior in females are undoubtedly due to methodological differences. However, because of the extremely complex, cyclic nature of the female endocrine system, an effect of a single exogenous hormone on behavior cannot be taken as proof that that particular hormone regulates the behavior in normal intact animals. First, as mentioned previously, synergistic actions among hormones are undoubtedly important. Second, the observed results may be indirect effects mediated by the secretion of some other hormone. For example, Christian’s (1971) report that adrenal weight in both male and female P. leucopus was above average during the breeding season suggests a possible influence of the reproductive hormones on adrenal function. Altered adrenal function, in turn, may be an important factor in the modification of agonistic behavior (cf. Edwards and Rowe, 1975). Also, Chen and Meites (1970) and Kalra, Fawcett, Krulich, and McCann (1973) found that estrogen, progesterone, and testosterone stimulated prolactin secretion in rats. Therefore, the effects we observed might also be due to stimulated prolactin secretion. Wise (1974) examined aggression during various reproductive stages of female hamsters and found that changes in aggression corresponded to changes in pituitary prolactin levels (Kent, 1968), including times when the ovaries are quiescent (Greenwald, 1965)and facilitation of aggression by ovarian hormones is unlikely. Noirot, Goyens, and Buhot (1975) reported patterns of aggression in pregnant and pseudopregnant female mice which correspond to increasing progesterone (Murr, Stabenfeldt, Bradford, and Geschwind, 1974) and prolactin (Murr, Bradford, and Geschwind, 1974) levels. Lactat-
38
GLEASON, MICHAEL,
AND CHRISTIAN
ing mice also exhibit patterns of aggression (Gandelman, 1972; St. John and Corning, 1973; Svare and Gandelman, 1973) which are similar to changes in circulating levels of prolactin in lactating mice (Sinha, Selby, and VanderLaan, 1974). This experiment has shown that gonadal steroids can affect the agonistic behavior of female P. leucopus. Further investigation is clearly necessary, however, to clarify the role of other hormones and hormone interactions in behavioral regulation in this species. REFERENCES Burt, W. H. (1940). Territorial behavior and populations of some small mammals in southern Michigan. Misc. Pub/. Mus. Zoo/. Univ. Mich. 45, 58~. Chen, C. L., and Meites, J. (1970). Effects of estrogen and progesterone on serum and pituitary prolactin levels in ovariectomized rats. Endocrinology 86, 503-505. Christian, J. J. (1971). Population density and reproductive efficiency. Biol. Reprod. 4,248. Edwards, D. A., and Rowe, F. A. (1975). Neural and endocrine control of aggressive behavior. In B. E. Eleftheriou and R. L. Sprott (Eds.), Hormonal Correlates of Behavior, Vol. 1. Plenum, New York. Gandelman, R. (1972). Mice: Postpartum aggression elicited by the presence of an intruder. Horm.
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Greenwald, G. S. (1%5). Histologic transformation of the ovary of the lactating hamster. Endocrinology
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Grelk, D. F., Papson, B. A., Cole, J. E., and Rowe, F. A. (1974). The influence of caging conditions and hormone treatments on fighting in male and female hamsters. Horm. Behav.
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Kalra, P. S., Fawcett, C. P., Krulich, L., and McCann, S. M. (1973). The effects of gonadal steroids on plasma gonadotropins and prolactin in the rat. Endocrino/ogy 92, 12561268. Kent, G. C. (1968). Physiology and reproduction. In R. A. Hoffman, P. F. Robinson, and H. Magalhaes (Eds.), The Golden Hamster: Its Biology and Use in Medical Research. Iowa Univ. Press, Ames. Kislak, J. W., and Beach, F. A. (1955). Inhibition of aggressiveness by ovarian hormones. Endocrinology
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Leshner, A. I., and Moyer, J. A. (1975). Androgens and agonistic behavior of mice: Relevance to aggression and irrelevance to avoidance-of-attack. Physiol. Behav. 15, 695-699. Leshner, A. I., Moyer, J. A., and Walker, W. A. (1975). Pituitary-adrenocortical activity and avoidance-of-attack in mice. Physiol Behav. 15, 689-693. Metzgar, L. H. (1971). Behavioral population regulation in the woodmouse, Peromyscus leucopus.
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Murie, J. 0. (1971). Dominance relationships between Peromyscus and Microtus in captivity. Amer. Midl. Natur. 86, 229-230. Murr, S. M., Bradford, G. E., and Geschwind, I. I. (1974). Plasma luteinizing hormone, follicle stimulating hormone, and prolactin during pregnancy in the mouse. Endocrinology
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Murr, S. M., Stabenfeldt, G. H., Bradford, G. E., and Geschwind, I. 1. (1974). Plasma progesterone during pregnancy in the mouse. Endocrinology 94, 1209-1211. Nicholson, A. J. (1941). The homes and social habits of the wood mouse (P. leucopus noveboracensis) in southern Michigan. Amer. Midl. Natur. 25, 196-223.
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Noirot, E., Goyens, J., and Buhot, M. C. (197.5). Aggressive behavior of pregnant mice toward males. Horm. Behav. 6, 9-17. Payne, A. P., and Swanson, H. H. (1970). Agonistic behaviour between pairs of hamsters of the same and opposite sex in a neutral observation arena. Behaviour 36, 259-269. Payne, A. P., and Swanson, H. H. (1971a). Hormonal control of aggressive dominance in the female hamster. Physiol. Behav. 6, 355-357. Payne, A. P., and Swanson, H. H. (1971b). Hormonal modification of aggressive behavior between female golden hamsters. J. Endocrinol. 51, xvii-xviii. Payne, A. P., and Swanson, H. H., (1972). The effect of sex hormones on the aggressive behavior of the female golden hamster (Mesocricetus auratus Waterhouse). Anim. Behav.
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St. John, R. D., and Corning, P. A. (1973). Maternal aggression in mice. Behav. Biol. 9, 635-639.
Sheppe, W. A. (1%6a). Social behavior of the deermouse Peromyscus leucopus in the laboratory. Wasmann J. Biol. 24, 49-65. Sheppe, W. A. (1%6b). Determinants of homerange in the deer mouse, Peromyscus Ieucopus.
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Sinha, Y. N., Selby, F. W., and VanderLaan, W. P. (1974). Relationship of prolactin and growth hormone to mammary function during pregnancy and lactation in the C3H/ST mouse. J. Endocrinol. 61, 219-229. Snyder, D. P. (1956). Survival rates, longevity, and population fluctuations in the whitefooted mouse, P. leucopus, in southeastern Michigan. Misc. Publ. Mus. Zool. Univ. Mich. 95, 33~. Svare, B., and Gandelman, R. (1973). Postpartum aggression in mice: Experiential and environmental factors. Horm. Behav. 4, 323-334. Tiefer, L. (1970). Gonadal hormones and mating behavior in the golden hamster. Horm. Behav.
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Vandenbergh, J. G. (1971). The effects of gonadal hormones on the aggressive behaviour of adult golden hamsters (Mesocricetus auratus). Anim. Behav. 19, 589-594. Wise, D. A. (1974). Aggression in the female golden hamster: Effects of reproductive state and social isolation. Horm. Behav. 5, 235-250.
