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Agonistic behavior between pregnant mice and male intruders
BEHAVIORAL AND NEURAL BIOLOGY31, 236--241 (1981)
BRIEF REPORT Agonistic Behavior between Pregnant Mice and Male Intruders CYNTHIA HEDRICKS 1'2 AND CRAIG E . DANIELS 1
Department of Psychology, University of Hartford, West Hartford, Connecticut 06117 Female mice in three reproductive states (pregnant, pseudopregnant, or natural estrous cycling) encountered a male intruder in the female's home cage. Although females and males exhibited both aggressive and submissive behaviors, females exhibited significantly more aggressive and less submissive behaviors than males. In addition, late gestational females displayed greatest frequency and duration of aggressive behaviors. The possible influences of the hormones of pregnancy and the home cage advantage of the female are discussed.
In a forced-encounter paradigm, wild-reared male mice consistently fight and chase strange males who enter their home cage area. Females, however, rarely exhibit these aggressive behavior patterns toward strang_ers. When these behaviors do occur in females, they usually appear during late gestation and/or lactation. Female Mus musculus L., Peromyscus maniculatus gambelii, and Peromyscus californicus parasit:~cus were aggressive toward strange males and females when pregnant, and often excluded the mate from the nest just prior to parturition (Eisenberg, 1962; Crowcroft & Rowe, 1963). The present study investigated a possible relationship between hormones and agonistic behavior (aggression and submission) between pregnant laboratory mice and a male intruder. In order to correlate observed behaviors with hormonal influences, testing was conducted on gestation days associated with particular hormonal fluctuations (i.e., Days 6, 12, and 18). Implantation is completed by Day 6, and the corpora lutea of This research was submitted to C.E.D. as partial requirement for the Master of Arts in Experimental Psychology at the University of Hartford. The authors wish to thank Ms. Cathy Daly for her invaluable assistance on the project, as well as the members of C.H.'s lab who were kind enough to read an earlier version of the paper. 2 Requests for reprints should be sent to Committee on Biopsychoiogy, University of Chicago, 5712 South Ingleside Ave., Chicago, Ill. 60637.
0163-1047/81/020236-06502.00/0 Copyright© 1981by AcademicPress, Inc. All fightsof reproductionin any formreserved.
236
AGONISTIC BEHAVIOR IN PREGNANTMICE
237
pregnancy become histologically demonstrable. Pseudopregnancy terminates by Days 10-12 while testosterone, progesterone, and estradiol levels continue to rise in the pregnant female (Barkley, Geschwind, & Bradford, 1979). On Day 18, just prior to parturition, there is typically a sudden drop in progesterone and a second peak in testosterone and estradiol (Barkley et al., 1979). Agonistic behaviors were recorded for both the female and the male intruder since it was felt that both animals' behavior would be influenced by the other, as suggested by Leshner (1975) in his hormone/agonistic behavior model. Also, repeated encounters between pregnant females and male intruders have yielded discrepant findings in two different studies of female aggression during pregnancy (Noirot, Goyens, & Buhot, 1975; Svare & Gandelman, 1976). The number of encounters was limited to one per animal in the present study. Mice from the Charles River CF-1 outbred albino strain were used. At weaning (Day 26) three males and three females were randomly selected from 32 litters and housed with same-sex littermates in opaque plastic cages, 28 x 13 x 15 cm high. The cage floors were covered with wood shavings. Food and water were available ad libitum, and a 12-hr L/D schedule was maintained (lights off from 2000-0800 hr). On Day 45 animals were housed individually. Females were randomly assigned to be tested in one of three reproductive states: Pregnant (P), n -- 30; Natural Estrous Cycling (C), n = 30; or Pseudopregnant (PP), n = 20. A male intruder was assigned to each female at this time in such a way that no female ever encountered a littermate as an intruder. Daily vaginal smears were taken from females at 1000 hr in order to determine estrous stage. When a female displayed a proestrous smear she received one of the following exposures to a colony male from 2000 to 0800 hr: In the Pregnant group, asexually experienced, intact male was placed in her home cage; in the Cycling group, a sexually experienced, intact male was placed on one side of a mesh barrier in her home cage; in the Pseudopregnant group, a vasectomized male with prior sexual experience was placed in her home cage. At 0800 hr the next day, males were returned to their colony cages. Females were checked for the appearance of the vaginal plug and placed into the testing cage (same dimensions as home cage). Cotton nesting material was provided for all females. Females in each group were then randomly assigned to be presented with a male intruder either 6, 12, or 18 days following the above exposure to the colony male. At 1900 hr on the appropriate day, the animals were transported in their home cages to the test room. The preassigned male was then placed into the female's home cage, at the opposite end from Where the female was located, and a 3-min encounter between the dyad was videotaped. There were a total of 10 forced encounters between Pregnant female
238
HEDRICKS AND DANIELS
and male intruder dyads on each of the 3 test days. In the Cycling group, there were 10 forced encounters on Days 6 and 18 and, 9 on day 12. Seventeen of the twenty females in the Pseudopregnant control group became pseudopregnant, resulting in a total of 8 forced encounters on Day 6, and 9 on Day 12. Since pseudopregnancy lasted approximately 12 days in these animals, no encounters occurred on Day 18. Males and females were scored separately for first approach, aggression, submission, and exploration of the test cage. Agonistic behaviors were scored following the criteria established by Grant and Mackintosh (1963) and Scott (1966). The criterion employed for scoring sequential aggressive or submissive episodes was that at least 4 sec had elapsed since the previous aggressive/submissive behavior. Females initiated more first approaches (F(I, 136) = 26.96, p < .0001); traveling a mean distance of 11.86 cm (SD = 6.10) compared to the males' 5.74 cm (SD = 6.83). Figure 1 shows the mean number of aggressive and submissive behaviors exhibited by both sexes in each condition. Over all conditions females were more aggressive than males (F(1, 136) = 55.43, p < .0001); however, the Reproductive State x Day of Testing interaction was also statistically significant (F(3, 136) = 5.24, p < .002). Female and male dyads tested in the Pregnant group on Day 18 displayed more aggression than dyads tested in any other condition (p < .05, Scheff6 test). Males were more submissive than females (F(I, 136) = 5.97, p < .01) and, as with aggression, the Reproductive State x Day of Testing interaction Was significant (F(3, 136) = 4.67, p < .01); dyads tested in the Pregnant group on Day 18 displayed more submissive behavior than all other dyads except those in the Pregnant group tested on Day 12 (p < .05). Those dyads in the Pregnant group tested on Day 12 were more submissive than dyads tested on Day 6 (p < .05). The three-way interaction (Sex x Reproductive State × Day of Testing) for duration of aggressive behaviors shown in Fig. 2 was also highly significant (F(3,136) = 4.50,p < .005). Pregnant females tested on Day 18 of gestation spent the greatest percentage of total test time engaged in aggressive behaviors (p < .05). Males spent more time exploring the test cage (F(I, 136) = 208.00, p < .0001); spending a mean amount of 70.72 (SD = 19.66) sec exploring, compared to the females' 26.17 (SD = 21.17) sec. Dyads tested on Days 6, 12, and 18 all differed significantly in amount of time spent exploring (F(2, 136) = 11.25, p < .0001); with means (SD) of 48.56 (29.12), 56.30 (30.38), and 37.29 (29.54) sec, respectively. All females exhibited agonistic behaviors classically attributed to their male conspecifics. Regardless of their reproductive condition, females were significantly more aggressive--attacking, biting, and pushing the male intruder. Although submissive behaviors in females were also ob-
239
AGONISTIC BEHAVIOR IN PREGNANT MICE
3O
Aggression o~"
2o
| E m ~
1(}
p
c
Day
o~
30r
~"
2o
~,
lO
pp
p
c
Day
6
pp 12
p
c
Day
18
p Day
18
Submission
p
c
Day
pp
6
p
c
Day
pp 12
c
FIG. 1. Total mean frequency of aggressive and submissive behaviors for both sexes in all groups. Testing took place on either Day 6, 12, or 18 of gestation. Reproductive state of female varied (P = Pregnant; C = Cycling; PP = Pseudopregnant). Female behaviors are depicted in the shaded portion of the bars; male behaviors are represented in the unshaded portion.
served, in no condition were females significantly more submissive than males. In contrast, male intruders were consistently more submissive than the females. A necessary condition for females to display the male patterns of aggression may be intrusion by a stranger into the proximity of the nest area, a familiar surrounding for the female. Ethological and laboratory studies of mice have found that a resident (male) is almost always successful in driving an intruder from his home range area (Eisenberg, 1962). In the present study, there were two findings which suggested that familiarity with the test situation may have been partially responsible for the sex differences in agonistic responding. First, male intruders spent 39% of the 3-min test exploring the test cage, compared to the females' 15% (p < .001). Second, resident females initiated significantly more first approaches to the intruder (p < .0001).
240
HEDRICKS AND DANIELS 30
"~ 20
[]
pregnant ~(~
]
cycling (~Q
•
pseudopregnant Q~)
,T
,
P
c Day 6
PP
P
e PO Day 12
P c Day 18
FIG. 2. Percentage of female aggression during the 180-sectest period. Letter designations are the same as in FIG. 1. Time spent being aggressive by males was not significant.
Of particular interest was the significant increase in the frequency of agonistic behaviors by the Pregnant group tested late in gestation (Day 18). Pregnant females in late gestation also spent the greatest percentage of total test time engaged in aggressive behaviors. It is suggested that elevated testosterone levels may be responsible for this increased frequency and persistence of aggression. Although testosterone and estrogen levels are high toward the end of gestation, research on ovariectomized mice implicates testosterone but not estrogen treatment as causing increased aggressiveness (Barkley & Goldman, 1978). In addition, testosterone has been related to persistence in social and searching behaviors, and binocular fixation on a novel stimulus in the home cage (i.e., in chicks; Andrew, 1975). The increased intensity and duration of aggression displayed by the females may have had a positive effect on submissiveness in the males. Agonistic responding by both sexes may also h a v e been influenced by prior female/male encounters during which the female had a similar hormonal milieu, e.g., testosterone levels peak during proestrus (Dupon & Kim, 1973). In the present study no behavioral observations were made during mating, or proestrus; however, others have found an increase in female aggression toward a male intruder during proestrus and metestrus (Hyde & Sawyer, 1977). These speculations, which are consistent with outcomes of dyadic encounters predicted by L e s h n e r ' s (1975) model of hormones and agonistic behavior, remain to be empirically tested.
REFERENCES Andrew, R. J. (1975). Effects of testosterone on the behaviour of the domestic chick. II. Effects present in both sexes. Animal Behaviour, 23, 156-168. Barkley, M, S., Geschwind, I. I., & Bradford, G. E. (1979). The gestational pattern of estradiol, testosterone and progesterone secretion in selected strains of mice. Biology of Reproduction, 20, 733-738.
AGONISTIC BEHAVIOR IN PREGNANT MICE
241
Barkley, M. S., & Goldman, B. (1978). Studies on opponent status and steroid mediation of aggression in female mice. Behavioral Biology, 23, 118-123. Crowcroft, P., & Rowe, F. P. (1963). Social organization and territorial behaviour in the wild house mouse (Mus musculus L.). Proceedings of the Zoological Society of London, 140, 517-531. Dupon, C., & Kim, M, H. (I 973). Peripheral plasma levels of testosterone, androstenedione, and oestradiol during the rat oestrous cycle. Journal of Endocrinology, 59, 653-654. Eisenberg, J. F. (1962). Studies in the behavior ofPeromyscus maniculatus gambelii and Peromyscus californicus parasiticus. Behaviour, 19, 177-207. Grant, E. C., & Mackintosh, J. H. (1963). A comparison of the social postures of some common laboratory rodents. Behaviour, 21, 246-259. Hyde, J. S., & Sawyer, T. F. (1977). Estrous cycle fluctuations in aggressiveness of house mice. Hormones and Behavior, 9, 290-295. Leshner, A. I. (1975). A model of hormones and agonistic behavior. Physiology and Behavior, 15, 225-235. Noirot, E., Goyens, J., & Buhot, M.-C. (1975). Aggressive behavior of pregnant mice towards males. Hormones and Behavior, 6, 9-17. Scott, J. P. (1966). Agonistic behavior of mice and rats: A review. American Zoologist, 6, 683-701. Svare, B., & Gandelman, R. (1976). A longitudinal analysis of maternal aggression in Rockland-Swiss albino mice. Developmental Psychobiology, 9, 437-446.
BRIEF REPORT Agonistic Behavior between Pregnant Mice and Male Intruders CYNTHIA HEDRICKS 1'2 AND CRAIG E . DANIELS 1
Department of Psychology, University of Hartford, West Hartford, Connecticut 06117 Female mice in three reproductive states (pregnant, pseudopregnant, or natural estrous cycling) encountered a male intruder in the female's home cage. Although females and males exhibited both aggressive and submissive behaviors, females exhibited significantly more aggressive and less submissive behaviors than males. In addition, late gestational females displayed greatest frequency and duration of aggressive behaviors. The possible influences of the hormones of pregnancy and the home cage advantage of the female are discussed.
In a forced-encounter paradigm, wild-reared male mice consistently fight and chase strange males who enter their home cage area. Females, however, rarely exhibit these aggressive behavior patterns toward strang_ers. When these behaviors do occur in females, they usually appear during late gestation and/or lactation. Female Mus musculus L., Peromyscus maniculatus gambelii, and Peromyscus californicus parasit:~cus were aggressive toward strange males and females when pregnant, and often excluded the mate from the nest just prior to parturition (Eisenberg, 1962; Crowcroft & Rowe, 1963). The present study investigated a possible relationship between hormones and agonistic behavior (aggression and submission) between pregnant laboratory mice and a male intruder. In order to correlate observed behaviors with hormonal influences, testing was conducted on gestation days associated with particular hormonal fluctuations (i.e., Days 6, 12, and 18). Implantation is completed by Day 6, and the corpora lutea of This research was submitted to C.E.D. as partial requirement for the Master of Arts in Experimental Psychology at the University of Hartford. The authors wish to thank Ms. Cathy Daly for her invaluable assistance on the project, as well as the members of C.H.'s lab who were kind enough to read an earlier version of the paper. 2 Requests for reprints should be sent to Committee on Biopsychoiogy, University of Chicago, 5712 South Ingleside Ave., Chicago, Ill. 60637.
0163-1047/81/020236-06502.00/0 Copyright© 1981by AcademicPress, Inc. All fightsof reproductionin any formreserved.
236
AGONISTIC BEHAVIOR IN PREGNANTMICE
237
pregnancy become histologically demonstrable. Pseudopregnancy terminates by Days 10-12 while testosterone, progesterone, and estradiol levels continue to rise in the pregnant female (Barkley, Geschwind, & Bradford, 1979). On Day 18, just prior to parturition, there is typically a sudden drop in progesterone and a second peak in testosterone and estradiol (Barkley et al., 1979). Agonistic behaviors were recorded for both the female and the male intruder since it was felt that both animals' behavior would be influenced by the other, as suggested by Leshner (1975) in his hormone/agonistic behavior model. Also, repeated encounters between pregnant females and male intruders have yielded discrepant findings in two different studies of female aggression during pregnancy (Noirot, Goyens, & Buhot, 1975; Svare & Gandelman, 1976). The number of encounters was limited to one per animal in the present study. Mice from the Charles River CF-1 outbred albino strain were used. At weaning (Day 26) three males and three females were randomly selected from 32 litters and housed with same-sex littermates in opaque plastic cages, 28 x 13 x 15 cm high. The cage floors were covered with wood shavings. Food and water were available ad libitum, and a 12-hr L/D schedule was maintained (lights off from 2000-0800 hr). On Day 45 animals were housed individually. Females were randomly assigned to be tested in one of three reproductive states: Pregnant (P), n -- 30; Natural Estrous Cycling (C), n = 30; or Pseudopregnant (PP), n = 20. A male intruder was assigned to each female at this time in such a way that no female ever encountered a littermate as an intruder. Daily vaginal smears were taken from females at 1000 hr in order to determine estrous stage. When a female displayed a proestrous smear she received one of the following exposures to a colony male from 2000 to 0800 hr: In the Pregnant group, asexually experienced, intact male was placed in her home cage; in the Cycling group, a sexually experienced, intact male was placed on one side of a mesh barrier in her home cage; in the Pseudopregnant group, a vasectomized male with prior sexual experience was placed in her home cage. At 0800 hr the next day, males were returned to their colony cages. Females were checked for the appearance of the vaginal plug and placed into the testing cage (same dimensions as home cage). Cotton nesting material was provided for all females. Females in each group were then randomly assigned to be presented with a male intruder either 6, 12, or 18 days following the above exposure to the colony male. At 1900 hr on the appropriate day, the animals were transported in their home cages to the test room. The preassigned male was then placed into the female's home cage, at the opposite end from Where the female was located, and a 3-min encounter between the dyad was videotaped. There were a total of 10 forced encounters between Pregnant female
238
HEDRICKS AND DANIELS
and male intruder dyads on each of the 3 test days. In the Cycling group, there were 10 forced encounters on Days 6 and 18 and, 9 on day 12. Seventeen of the twenty females in the Pseudopregnant control group became pseudopregnant, resulting in a total of 8 forced encounters on Day 6, and 9 on Day 12. Since pseudopregnancy lasted approximately 12 days in these animals, no encounters occurred on Day 18. Males and females were scored separately for first approach, aggression, submission, and exploration of the test cage. Agonistic behaviors were scored following the criteria established by Grant and Mackintosh (1963) and Scott (1966). The criterion employed for scoring sequential aggressive or submissive episodes was that at least 4 sec had elapsed since the previous aggressive/submissive behavior. Females initiated more first approaches (F(I, 136) = 26.96, p < .0001); traveling a mean distance of 11.86 cm (SD = 6.10) compared to the males' 5.74 cm (SD = 6.83). Figure 1 shows the mean number of aggressive and submissive behaviors exhibited by both sexes in each condition. Over all conditions females were more aggressive than males (F(1, 136) = 55.43, p < .0001); however, the Reproductive State x Day of Testing interaction was also statistically significant (F(3, 136) = 5.24, p < .002). Female and male dyads tested in the Pregnant group on Day 18 displayed more aggression than dyads tested in any other condition (p < .05, Scheff6 test). Males were more submissive than females (F(I, 136) = 5.97, p < .01) and, as with aggression, the Reproductive State x Day of Testing interaction Was significant (F(3, 136) = 4.67, p < .01); dyads tested in the Pregnant group on Day 18 displayed more submissive behavior than all other dyads except those in the Pregnant group tested on Day 12 (p < .05). Those dyads in the Pregnant group tested on Day 12 were more submissive than dyads tested on Day 6 (p < .05). The three-way interaction (Sex x Reproductive State × Day of Testing) for duration of aggressive behaviors shown in Fig. 2 was also highly significant (F(3,136) = 4.50,p < .005). Pregnant females tested on Day 18 of gestation spent the greatest percentage of total test time engaged in aggressive behaviors (p < .05). Males spent more time exploring the test cage (F(I, 136) = 208.00, p < .0001); spending a mean amount of 70.72 (SD = 19.66) sec exploring, compared to the females' 26.17 (SD = 21.17) sec. Dyads tested on Days 6, 12, and 18 all differed significantly in amount of time spent exploring (F(2, 136) = 11.25, p < .0001); with means (SD) of 48.56 (29.12), 56.30 (30.38), and 37.29 (29.54) sec, respectively. All females exhibited agonistic behaviors classically attributed to their male conspecifics. Regardless of their reproductive condition, females were significantly more aggressive--attacking, biting, and pushing the male intruder. Although submissive behaviors in females were also ob-
239
AGONISTIC BEHAVIOR IN PREGNANT MICE
3O
Aggression o~"
2o
| E m ~
1(}
p
c
Day
o~
30r
~"
2o
~,
lO
pp
p
c
Day
6
pp 12
p
c
Day
18
p Day
18
Submission
p
c
Day
pp
6
p
c
Day
pp 12
c
FIG. 1. Total mean frequency of aggressive and submissive behaviors for both sexes in all groups. Testing took place on either Day 6, 12, or 18 of gestation. Reproductive state of female varied (P = Pregnant; C = Cycling; PP = Pseudopregnant). Female behaviors are depicted in the shaded portion of the bars; male behaviors are represented in the unshaded portion.
served, in no condition were females significantly more submissive than males. In contrast, male intruders were consistently more submissive than the females. A necessary condition for females to display the male patterns of aggression may be intrusion by a stranger into the proximity of the nest area, a familiar surrounding for the female. Ethological and laboratory studies of mice have found that a resident (male) is almost always successful in driving an intruder from his home range area (Eisenberg, 1962). In the present study, there were two findings which suggested that familiarity with the test situation may have been partially responsible for the sex differences in agonistic responding. First, male intruders spent 39% of the 3-min test exploring the test cage, compared to the females' 15% (p < .001). Second, resident females initiated significantly more first approaches to the intruder (p < .0001).
240
HEDRICKS AND DANIELS 30
"~ 20
[]
pregnant ~(~
]
cycling (~Q
•
pseudopregnant Q~)
,T
,
P
c Day 6
PP
P
e PO Day 12
P c Day 18
FIG. 2. Percentage of female aggression during the 180-sectest period. Letter designations are the same as in FIG. 1. Time spent being aggressive by males was not significant.
Of particular interest was the significant increase in the frequency of agonistic behaviors by the Pregnant group tested late in gestation (Day 18). Pregnant females in late gestation also spent the greatest percentage of total test time engaged in aggressive behaviors. It is suggested that elevated testosterone levels may be responsible for this increased frequency and persistence of aggression. Although testosterone and estrogen levels are high toward the end of gestation, research on ovariectomized mice implicates testosterone but not estrogen treatment as causing increased aggressiveness (Barkley & Goldman, 1978). In addition, testosterone has been related to persistence in social and searching behaviors, and binocular fixation on a novel stimulus in the home cage (i.e., in chicks; Andrew, 1975). The increased intensity and duration of aggression displayed by the females may have had a positive effect on submissiveness in the males. Agonistic responding by both sexes may also h a v e been influenced by prior female/male encounters during which the female had a similar hormonal milieu, e.g., testosterone levels peak during proestrus (Dupon & Kim, 1973). In the present study no behavioral observations were made during mating, or proestrus; however, others have found an increase in female aggression toward a male intruder during proestrus and metestrus (Hyde & Sawyer, 1977). These speculations, which are consistent with outcomes of dyadic encounters predicted by L e s h n e r ' s (1975) model of hormones and agonistic behavior, remain to be empirically tested.
REFERENCES Andrew, R. J. (1975). Effects of testosterone on the behaviour of the domestic chick. II. Effects present in both sexes. Animal Behaviour, 23, 156-168. Barkley, M, S., Geschwind, I. I., & Bradford, G. E. (1979). The gestational pattern of estradiol, testosterone and progesterone secretion in selected strains of mice. Biology of Reproduction, 20, 733-738.
AGONISTIC BEHAVIOR IN PREGNANT MICE
241
Barkley, M. S., & Goldman, B. (1978). Studies on opponent status and steroid mediation of aggression in female mice. Behavioral Biology, 23, 118-123. Crowcroft, P., & Rowe, F. P. (1963). Social organization and territorial behaviour in the wild house mouse (Mus musculus L.). Proceedings of the Zoological Society of London, 140, 517-531. Dupon, C., & Kim, M, H. (I 973). Peripheral plasma levels of testosterone, androstenedione, and oestradiol during the rat oestrous cycle. Journal of Endocrinology, 59, 653-654. Eisenberg, J. F. (1962). Studies in the behavior ofPeromyscus maniculatus gambelii and Peromyscus californicus parasiticus. Behaviour, 19, 177-207. Grant, E. C., & Mackintosh, J. H. (1963). A comparison of the social postures of some common laboratory rodents. Behaviour, 21, 246-259. Hyde, J. S., & Sawyer, T. F. (1977). Estrous cycle fluctuations in aggressiveness of house mice. Hormones and Behavior, 9, 290-295. Leshner, A. I. (1975). A model of hormones and agonistic behavior. Physiology and Behavior, 15, 225-235. Noirot, E., Goyens, J., & Buhot, M.-C. (1975). Aggressive behavior of pregnant mice towards males. Hormones and Behavior, 6, 9-17. Scott, J. P. (1966). Agonistic behavior of mice and rats: A review. American Zoologist, 6, 683-701. Svare, B., & Gandelman, R. (1976). A longitudinal analysis of maternal aggression in Rockland-Swiss albino mice. Developmental Psychobiology, 9, 437-446.