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Interaction of genotype and exogenous neonatal estrogen: Aggression in female mice
Physiology and Behavior, Vol. 10, pp. 181-183. Brain Research Publications Inc., 1973. Printed in the U.S.A.
Interaction of Genotype and Exogenous Neonatal Estrogen: Aggression in Female Mice ' J A C K R. V A L E z , D O N A L D R A Y A N D C A R O L A. V A L E
Bowling Green State University, Bowling Green, Ohio 43402
(Received 1 May 1972) VALE, J. R., D. RAY AND C. A. VALE. Interaction o f genotype and exogenous neonatal estrogen: aggression in female mice. PHYSIOL. BEHAV. 10(2) 181-183, 1973. Defensive aggression in neonataUy estrogen treated mice was increased in females of an inbred strain wherein the males are highly aggressive, but there was no change in females from strains wherein the males are relatively passive. Such genotype X treatment interaction suggests that genotype is a potentially important variable for study of the effects of early sex hormones. The interaction may be due to strain differences in relevant genes activated during the period of maximum organizational effects of sex hormone. Mouse genotype Estrogenization
Neonatal estrogen Interaction Agonistic behavior Aggression Species specific behavior Sex hormones Genes Inbred strains
A N U M B E R of studies [ 1,21 have shown that female mice treated with testosterone p r o p i o n a t e (TP) during the early neonatal period are masculinized and exhibit an increased a m o u n t of aggression as adults. F e m a l e mice are normally unaggressive. However, the effects of variation in g e n o t y p e upon this p h e n o m e n o n are only now being reported. Since male mice of different g e n o t y p e s differ in their propensity for aggression [ 6, 7, 11 ], and since sex h o r m o n e s have been shown to regulate gene activity in peripheral tissue [5,9], study of the simultaneous variation of g e n o t y p e and exogenous sex h o r m o n e administered during the early neonatal period would seem to be a logical extension of previous work. It may be that the organizational effects of sex h o r m o n e s in brain are due to the activation or repression of genes. Inbred strains provide suitable material with which to begin work u p o n this hypothesis, since b e t w e e n strains there are b o t h genetic differences and differences in male behavior, while within strains most genes are shared b e t w e e n males and females but there is sexual dimorphism in behavior. In a previous report [ 1 0 ] , we found that females of different genotypes treated neonatally with TP differ in the extent of their adult aggressive responses. In tests of defensive aggression, females from a strain wherein the males are aggressive were m a d e highly aggressive by neonatal TP, whereas females from two strains wherein the males are m u c h less aggressive were largely unaffected. Since b o t h androgens and estrogens administered neonatally masculinize female reproductive physiology [4] and estrogen alters female sexual receptivity [ 12] and aggression in some g e n o t y p e s [1,3], it is of interest to study the effects o f neonatal estrogen when g e n o t y p e is varied
systematically. We report here u p o n the effects of neonatally administered estradiol benzoate (EB) on adult aggressiveness in females from three strains of mice. METHOD AND MATERIALS Procedures were the same as used when androgen was administered [10] with the e x c e p t i o n of h o r m o n e and dose. A, BALB/c, and C57BL/6 mice [8] were mated within strains and whole litters injected subcutaneously on the 3rd day of life with either 0.02 ml peanut oil containing 0.5 mg EB or an equal v o l u m e of oil alone. Litters were weaned at 30 days and the individuals isolated in plastic cages ( 2 8 . 5 x 1 8 . 5 x 1 2 cm) until 7 5 - 9 0 days, when testing began. The light cycle throughout was 7 a.m. to 7 p.m. - red light; 7 p.m. to 7 a.m. - white light. All observations were made under red light, during the animals' normally active period. T w e n t y females of each g e n o t y p e were used in each t r e a t m e n t c o n d i t i o n (thus, N=120). Aggression tests were made during the course of 7 days of tests for agonistic behavior and male and female sex behavior. The tests described here are for defensive aggression, i.e., aggression in reaction to the actions of another animal. In the dangler test, untreated males of the A strain were held by the tail with forceps and dragged gently along the b o t t o m o f the female's cage. Occasionally the female was gently b u m p e d by the stimulus male. The latency and n u m b e r of the female's attacks, and whether she squeaked or ran away during the first 3 rain period were recorded. The stimulus male was removed for 1 min, and then the procedure was repeated for 1 1/2 min, immediately
1Supported by USPHS Grant MH 17629 to the first author. Computing assistance was obtained from the Health Sciences Computing Facility, UCLA, sponsored by NIH Special Resources Grant RR-3. 2Reprint requests should be addressed to Jack R. Vale, Department of Psychology, University of California, Berkeley, California 94720. 181
182
VALti, RA5 AN I) VAIA;
following which tire male was allowed to run free m the female's cage for I 1/2 rain. These periods were designated 1st, 2nd, and free dangler sessions. The entire test was repeated over 3 consecutive days. For 2 consecutive days following the dangler test, each female was given an intramuscular injection of 0.02 mg EB in oil. Thirty-six hr after the 2nd injection she was injected with 0.2 mg progesterone, and 3 5 hr later a sexually vigorous C57BL/6 male was introduced into her cage. The latency and number of attacks against the male in a 1/2 hr period were recorded.
RESULTS Latency scores were transformed to reciprocals, in accordance with usual practice, in order to avoid skewing. Nonresponders were assigned a transformed score of zero. Thus, the larger the value reported, the faster the response. Reciprocal latencies and number of attacks for each dangler session (averaged over 3 days) and for the test with the male attempting to mate were subjected to a 3 x 2 (strains x treatments) analysis of variance, fixed effects model. Differences in latency and number of attacks in the dangler sessions were not significant, although treated BALB/c females were the only ones to respond. The effects of neonatal estrogen upon the proportion of animals squeaking and running away during the dangler test was genotype dependent, Squeaking was reduced only in BALB/c's (p<0.0007 by chi square analysis), and running away was similarly reduced in treated BALB/c's (p<0.05). Table 1 presents the mean reciprocal latency to attack and the mean number of attacks made against the male attempting to mate. Differences between strains, treatments, and the interaction were significant on latency (p<0.003; p<0.02; p <0.003) and number (p<0.01; 10<0.04; p<0.01). However, the effect is obviously also genotype dependent, since the statistical significance is derived from differences between treated BALB/c females and the others. Differences in the proportions of animals responding are significant for the BALB/c's (p<0.03 by Fisher's exact test).
I)ISCUSSI()N The present results support the conclusion ol our previous study that the effects of exogenous neonatal sex steroids upon aggression in female micc are genotype dependent. In that study it was also found that the treatment affected BALB/c to a much greater extent than A or C57BL/6 females. Normal BALB/c males have been found to be highly aggressive ira comparison with A and C57BL/6 males [6, 7, 11]. This provides indirect support for the proposition that the organizational effects of neonatal sex steroids may be due to gene activation or repression during this sensitive period. However, comparison of data from our previous and present studies indicates that neonatal administration of EB is less effective in producing defensive aggression than is TP. When TP was used, strain, treatment, and interaction effects on the 1st and 2nd dangler sessions were highly significant and due to the BALB/c females, which had mean numbers of attacks of 11.98 and 10.07, respectively. In addition, 13/20 treated BALB/c's attacked the dangler. Similarly, the mean number of attacks made against the male attempting to mate by the androgenized BALB/c females was 4 times that of the estrogenized BALB/c's and 17/20 of the androgenized BALB/c's attacked, although statistical significance was obtained with both hormones. This difference in the relative effects of the two horlnones is in agreement with that reported for spontaneous aggression in C57BL/6 females [1 ], where tl~e ratio of fights/encounters in estrogenized pairs of females {5/42) was less than that of androgenized pairs {10/54). The proportion of estrogenized females attacking males (4/14) was similarly less than that of androgenized females (12/23) in the same study. (In contrast, another report [31 found that an attack occurred at least once in nearly equal percentages of neonatally estrogenized and androgenized female Swiss Webster mouse pairs gonadectoinized and given TP replacement, l Thus, while TP and EB masculinize the hypothalamic pituitary system controlling gonadotrophin secretion in females equally well I4]. our data and those of others suggest that the behavioral effects may be at least partially hormone specific, although consistent within genotypes.
TABLE 1 MEAN RECIPROCAL LATENCY TO ATTACK AND MEAN NUMBER OF ATTACKS MADE AGAINST THE MALE ATTEMPTING TO MATE*
Strain
Treatment
EB
Oil
Mean reciprocal latency to attack A BALB/C C57BL/6
0.00 0.05 0.00
0.00 0,00 0,00
Treatment
EB
Oil
Mean number of attacks 0.00(0) 5.76(6) 0.00(0)
0.00(0) 0.00(0) 0.00(0)
*Numbers of animals responding at least once are in parentheses.
G E N O T Y P E A N D N E O N A T A L E S T R O G E N IN MICE
183
REFERENCES 1. Bronson, F. H. and C. Desjardins. Aggression in adult mice: Modification by neonatal injections of gonadal hormones. Science 161: 7 0 5 - 7 0 6 , 1968. 2. Edwards, D. A. Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behav. 4: 333-338, 1969. 3. Edwards, D. A. and J. Herndon. Neonatal estrogen stimulation and aggressive behavior in female mice. Physiol. Behav. 5: 9 9 3 - 9 9 5 , 1970. 4. Gorski, R. A. and J. W. Wagner. Gonadal activity and sexual differentiation of the hypothalamus. Endocrinology 76: 2 2 6 - 2 3 9 , 1965. 5. Hamilton, T. H. Control by estrogen of genetic transcription and translation. Science 161: 6 4 9 - 6 6 1 , 1968. 6. Scott, J. P. and E. Fredericson. The causes of fighting in mice and rats. Physiol. Zool. 24: 273-309, 1951. 7. Southwick, C. H. and L. H. Clark. Interstrain differences in aggressive behavior and exploratory activity of inbred mice. Communs behav. BioL 1 : 4 5 - 5 9 , 1968.
8. Staats, J. The laboratory mouse. In: Biology of the Laboratory Mouse, edited by E. L. Green. New York: McGraw Hill, 1966, pp. 1-9. 9. Tata, J. R. Hormones and the synthesis and utilization of ribonucleic acids.Prog, nucL AcidRes. 5: 191-250, 1966. 10. Vale, J. R., D. Ray and C. A. Vale. Interaction of genotype and exogenous neonotal androgen: agonistic behavior in female mice. Behav. Biol. 7: 321-334, 1972. 11. Vale, J. R., C. A. Vale and J. P. Harley. Interaction of genotype and population number with regard to aggressive behavior, social grooming, and adrenal and gonadal weight in male mice. Cornmuns behav. Biol. 6: 209-221, 1971. 12. Whalen, R. E. and R. D. Nadler. Suppression of the development of female mating behavior by estrogen administered in infancy. Science 141: 273-274, 1963.
Interaction of Genotype and Exogenous Neonatal Estrogen: Aggression in Female Mice ' J A C K R. V A L E z , D O N A L D R A Y A N D C A R O L A. V A L E
Bowling Green State University, Bowling Green, Ohio 43402
(Received 1 May 1972) VALE, J. R., D. RAY AND C. A. VALE. Interaction o f genotype and exogenous neonatal estrogen: aggression in female mice. PHYSIOL. BEHAV. 10(2) 181-183, 1973. Defensive aggression in neonataUy estrogen treated mice was increased in females of an inbred strain wherein the males are highly aggressive, but there was no change in females from strains wherein the males are relatively passive. Such genotype X treatment interaction suggests that genotype is a potentially important variable for study of the effects of early sex hormones. The interaction may be due to strain differences in relevant genes activated during the period of maximum organizational effects of sex hormone. Mouse genotype Estrogenization
Neonatal estrogen Interaction Agonistic behavior Aggression Species specific behavior Sex hormones Genes Inbred strains
A N U M B E R of studies [ 1,21 have shown that female mice treated with testosterone p r o p i o n a t e (TP) during the early neonatal period are masculinized and exhibit an increased a m o u n t of aggression as adults. F e m a l e mice are normally unaggressive. However, the effects of variation in g e n o t y p e upon this p h e n o m e n o n are only now being reported. Since male mice of different g e n o t y p e s differ in their propensity for aggression [ 6, 7, 11 ], and since sex h o r m o n e s have been shown to regulate gene activity in peripheral tissue [5,9], study of the simultaneous variation of g e n o t y p e and exogenous sex h o r m o n e administered during the early neonatal period would seem to be a logical extension of previous work. It may be that the organizational effects of sex h o r m o n e s in brain are due to the activation or repression of genes. Inbred strains provide suitable material with which to begin work u p o n this hypothesis, since b e t w e e n strains there are b o t h genetic differences and differences in male behavior, while within strains most genes are shared b e t w e e n males and females but there is sexual dimorphism in behavior. In a previous report [ 1 0 ] , we found that females of different genotypes treated neonatally with TP differ in the extent of their adult aggressive responses. In tests of defensive aggression, females from a strain wherein the males are aggressive were m a d e highly aggressive by neonatal TP, whereas females from two strains wherein the males are m u c h less aggressive were largely unaffected. Since b o t h androgens and estrogens administered neonatally masculinize female reproductive physiology [4] and estrogen alters female sexual receptivity [ 12] and aggression in some g e n o t y p e s [1,3], it is of interest to study the effects o f neonatal estrogen when g e n o t y p e is varied
systematically. We report here u p o n the effects of neonatally administered estradiol benzoate (EB) on adult aggressiveness in females from three strains of mice. METHOD AND MATERIALS Procedures were the same as used when androgen was administered [10] with the e x c e p t i o n of h o r m o n e and dose. A, BALB/c, and C57BL/6 mice [8] were mated within strains and whole litters injected subcutaneously on the 3rd day of life with either 0.02 ml peanut oil containing 0.5 mg EB or an equal v o l u m e of oil alone. Litters were weaned at 30 days and the individuals isolated in plastic cages ( 2 8 . 5 x 1 8 . 5 x 1 2 cm) until 7 5 - 9 0 days, when testing began. The light cycle throughout was 7 a.m. to 7 p.m. - red light; 7 p.m. to 7 a.m. - white light. All observations were made under red light, during the animals' normally active period. T w e n t y females of each g e n o t y p e were used in each t r e a t m e n t c o n d i t i o n (thus, N=120). Aggression tests were made during the course of 7 days of tests for agonistic behavior and male and female sex behavior. The tests described here are for defensive aggression, i.e., aggression in reaction to the actions of another animal. In the dangler test, untreated males of the A strain were held by the tail with forceps and dragged gently along the b o t t o m o f the female's cage. Occasionally the female was gently b u m p e d by the stimulus male. The latency and n u m b e r of the female's attacks, and whether she squeaked or ran away during the first 3 rain period were recorded. The stimulus male was removed for 1 min, and then the procedure was repeated for 1 1/2 min, immediately
1Supported by USPHS Grant MH 17629 to the first author. Computing assistance was obtained from the Health Sciences Computing Facility, UCLA, sponsored by NIH Special Resources Grant RR-3. 2Reprint requests should be addressed to Jack R. Vale, Department of Psychology, University of California, Berkeley, California 94720. 181
182
VALti, RA5 AN I) VAIA;
following which tire male was allowed to run free m the female's cage for I 1/2 rain. These periods were designated 1st, 2nd, and free dangler sessions. The entire test was repeated over 3 consecutive days. For 2 consecutive days following the dangler test, each female was given an intramuscular injection of 0.02 mg EB in oil. Thirty-six hr after the 2nd injection she was injected with 0.2 mg progesterone, and 3 5 hr later a sexually vigorous C57BL/6 male was introduced into her cage. The latency and number of attacks against the male in a 1/2 hr period were recorded.
RESULTS Latency scores were transformed to reciprocals, in accordance with usual practice, in order to avoid skewing. Nonresponders were assigned a transformed score of zero. Thus, the larger the value reported, the faster the response. Reciprocal latencies and number of attacks for each dangler session (averaged over 3 days) and for the test with the male attempting to mate were subjected to a 3 x 2 (strains x treatments) analysis of variance, fixed effects model. Differences in latency and number of attacks in the dangler sessions were not significant, although treated BALB/c females were the only ones to respond. The effects of neonatal estrogen upon the proportion of animals squeaking and running away during the dangler test was genotype dependent, Squeaking was reduced only in BALB/c's (p<0.0007 by chi square analysis), and running away was similarly reduced in treated BALB/c's (p<0.05). Table 1 presents the mean reciprocal latency to attack and the mean number of attacks made against the male attempting to mate. Differences between strains, treatments, and the interaction were significant on latency (p<0.003; p<0.02; p <0.003) and number (p<0.01; 10<0.04; p<0.01). However, the effect is obviously also genotype dependent, since the statistical significance is derived from differences between treated BALB/c females and the others. Differences in the proportions of animals responding are significant for the BALB/c's (p<0.03 by Fisher's exact test).
I)ISCUSSI()N The present results support the conclusion ol our previous study that the effects of exogenous neonatal sex steroids upon aggression in female micc are genotype dependent. In that study it was also found that the treatment affected BALB/c to a much greater extent than A or C57BL/6 females. Normal BALB/c males have been found to be highly aggressive ira comparison with A and C57BL/6 males [6, 7, 11]. This provides indirect support for the proposition that the organizational effects of neonatal sex steroids may be due to gene activation or repression during this sensitive period. However, comparison of data from our previous and present studies indicates that neonatal administration of EB is less effective in producing defensive aggression than is TP. When TP was used, strain, treatment, and interaction effects on the 1st and 2nd dangler sessions were highly significant and due to the BALB/c females, which had mean numbers of attacks of 11.98 and 10.07, respectively. In addition, 13/20 treated BALB/c's attacked the dangler. Similarly, the mean number of attacks made against the male attempting to mate by the androgenized BALB/c females was 4 times that of the estrogenized BALB/c's and 17/20 of the androgenized BALB/c's attacked, although statistical significance was obtained with both hormones. This difference in the relative effects of the two horlnones is in agreement with that reported for spontaneous aggression in C57BL/6 females [1 ], where tl~e ratio of fights/encounters in estrogenized pairs of females {5/42) was less than that of androgenized pairs {10/54). The proportion of estrogenized females attacking males (4/14) was similarly less than that of androgenized females (12/23) in the same study. (In contrast, another report [31 found that an attack occurred at least once in nearly equal percentages of neonatally estrogenized and androgenized female Swiss Webster mouse pairs gonadectoinized and given TP replacement, l Thus, while TP and EB masculinize the hypothalamic pituitary system controlling gonadotrophin secretion in females equally well I4]. our data and those of others suggest that the behavioral effects may be at least partially hormone specific, although consistent within genotypes.
TABLE 1 MEAN RECIPROCAL LATENCY TO ATTACK AND MEAN NUMBER OF ATTACKS MADE AGAINST THE MALE ATTEMPTING TO MATE*
Strain
Treatment
EB
Oil
Mean reciprocal latency to attack A BALB/C C57BL/6
0.00 0.05 0.00
0.00 0,00 0,00
Treatment
EB
Oil
Mean number of attacks 0.00(0) 5.76(6) 0.00(0)
0.00(0) 0.00(0) 0.00(0)
*Numbers of animals responding at least once are in parentheses.
G E N O T Y P E A N D N E O N A T A L E S T R O G E N IN MICE
183
REFERENCES 1. Bronson, F. H. and C. Desjardins. Aggression in adult mice: Modification by neonatal injections of gonadal hormones. Science 161: 7 0 5 - 7 0 6 , 1968. 2. Edwards, D. A. Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behav. 4: 333-338, 1969. 3. Edwards, D. A. and J. Herndon. Neonatal estrogen stimulation and aggressive behavior in female mice. Physiol. Behav. 5: 9 9 3 - 9 9 5 , 1970. 4. Gorski, R. A. and J. W. Wagner. Gonadal activity and sexual differentiation of the hypothalamus. Endocrinology 76: 2 2 6 - 2 3 9 , 1965. 5. Hamilton, T. H. Control by estrogen of genetic transcription and translation. Science 161: 6 4 9 - 6 6 1 , 1968. 6. Scott, J. P. and E. Fredericson. The causes of fighting in mice and rats. Physiol. Zool. 24: 273-309, 1951. 7. Southwick, C. H. and L. H. Clark. Interstrain differences in aggressive behavior and exploratory activity of inbred mice. Communs behav. BioL 1 : 4 5 - 5 9 , 1968.
8. Staats, J. The laboratory mouse. In: Biology of the Laboratory Mouse, edited by E. L. Green. New York: McGraw Hill, 1966, pp. 1-9. 9. Tata, J. R. Hormones and the synthesis and utilization of ribonucleic acids.Prog, nucL AcidRes. 5: 191-250, 1966. 10. Vale, J. R., D. Ray and C. A. Vale. Interaction of genotype and exogenous neonotal androgen: agonistic behavior in female mice. Behav. Biol. 7: 321-334, 1972. 11. Vale, J. R., C. A. Vale and J. P. Harley. Interaction of genotype and population number with regard to aggressive behavior, social grooming, and adrenal and gonadal weight in male mice. Cornmuns behav. Biol. 6: 209-221, 1971. 12. Whalen, R. E. and R. D. Nadler. Suppression of the development of female mating behavior by estrogen administered in infancy. Science 141: 273-274, 1963.