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Post-neonatal androgenization and adult aggressive behavior in female mice
Physiology and Behavior. Vol. 5, pp. 465.-467. Pergamon Press, 1970. Printed in Great Britain
Post-Neonatal Androgenization and Adult Aggressive Behavior in Female Mice' D A V I D A. E D W A R D S
Department of Psychology, Emory University, Atlanta, Georgia, U.S.A. (Received 23 S e p t e m b e r 1969) EDWARDS, D. A. Post-neonatal androgenization and adult aggressive behavior in female mice. PHYSIOL. BEHAV. 5 (4) 465--467, 1970.--This experiment sought to determine whether chronic, long term administration of testosterone propionate (TP) to post-neonatal female mice would facilitate the androgen induced arousal of aggressive behavior in adulthood. Female mice were ovariectomized at 30 days of age and either administered TP daily for 20 days or a control injection of oil for 20 days. Forty-five days after the termination of the pretreatment, all mice were given TP and tested in pairs for fighting. Following androgen administration in adulthood 75 per cent of the pairs pretreated with TP in immaturity fought, but only 25 per cent of the pairs of females pretreated with oil in immaturity fought. It was concluded that the potential for androgen administered in infancy to induce masculinization of female mice with respect to androgen aroused aggressive behavior in adulthood exists for at least one month after birth. Mice
Androgen
Brain differentiation
Aggression
IT HAS BEEN known for some years that isolated male mice readily fight when paired. Castration of prepuberaily isolated male mice results in the failure of the mice to display aggressive behavior when a period of 25 or more days elapses between castration and testing: replacement therapy with exogenous androgen induces castrates to fight with other mice [1 ]. Thus, aggressive behavior in male mice is, at least partially, dependent upon androgen stimulation. In contrast to male mice, female mice rarely fight. Female mice do not even fight when administered large amounts of exogenous androgen [5]. Presuming that testosterone exerts its effects upon aggressive behavior through its action on central nervous system structures, these findings suggest that male and female mice possess functionally or structurally different neuroendocrine mechanisms relating to the control of aggression. In a recent series of experiments, Edwards [2, 3] showed that male mice and female mice given testosterone propionate (TP) on the day of birth displayed comparable levels of aggression when gonadectomized and tested for fighting following androgen administration in adulthood. Female mice treated only with oil at birth, failed to show aggression even when given large amounts of exogenous androgen in adulthood. Females given TP on the 10th day after birth showed more aggression than normal females, but less aggression than females given TP on the day of birth. These findings indicate that androgen stimulation of the neonatal female mouse masculinizes the female with respect to aggressiveness by causing or facilitating the differentiation of neuroendocrine mechanisms for aggression. Furthermore, the finding that females given TP 10 days after birth are less aggressive as
Development
adults than females given testosterone on the day of birth indicates the existence of a critical period for the androgen induced organization of mechanisms for aggression such that by the 10th day after birth the potential for such differentiation to occur is somewhat attenuated. While Edwards' findings indicate the existence of a critical period for the masculinization process in mice with respect to aggressive behavior, it should be recognized that the limits of the critical period are undoubtedly affected by the dose level of masculinizing androgen administered. Thus, Edwards' findings do not necessarily indicate that the potential for the androgen induced differentiation of hormone sensitive mechanisms for aggression is totally absent after the first few weeks after birth; rather, the results indicate that the capacity for a single injection of a given amount of androgen to facilitate aggressive behavior in the female is diminished by the 10th day after birth. It might be possible, for example, to masculinize female mice, with respect to aggressive behavior, much later in infancy or early adulthood if more extreme androgen manipulations were performed. This study reports the successful attempt to behaviorally masculinize female mice with respect to aggressive behavior by chronic androgen administration begun when the female mice were 30 days of age. METHOD
Subjects Sixty-four female Swiss-Webster mice (commercially obtained: Simonson Laboratories, Gilroy, California), 30
Wartial support was provided by a Pre-doctoral Research Fellowship from the USPHS (MH 30, 315-03), by grant HD 00893 from the National Institute of Child Health and Human Development to R. E. Whalen, by a research grant from Emory University, and by a Public Health Service Research Grant MH 17291 to D. A. Edwards. The testosterone used was generously supplied by Dr. Preston Perlman, Sebering Corporation, Bloomfield, New Jersey. 465
466
i!DWA
days old at the start of the experiment were raised and maintained under a reversed light-dark cycle with food and water ad lib.
t~.I ~F;
conducted during the first few hours of the dark phase of the lighting cycle. RESULTS
Procedure
As shown in Fig. 1, adult androgen administration lo animals in each of the experimental groups resulted in an increase in the proportion of pairs in each of the groups fighting. However, the effects of TP administration in adulthood upon fighting are much more striking for the group pretreated with testosterone in immaturity than for the group receiving control injections of oil during immaturity. In adulthood, 75 per cent of the pairs in the TP-female group fought at least once during the series of tests, but only 25 per cent of the pairs in the Oil-female group fought at least once. This difference is significant at beyond the 0.01 level (Chi-square = 8.0, df = 1). While very few pairs in either group fought at the lower hormone levels, a significantly greater proportion of TP-female pairs fought than Oil-female pairs at the 50 izg level. (Chi-square = 5.92, df = 1, p < 0.02). At the 100 izg and 500 t~g levels, TP-females fought more than Oil-females, and differences at each of these levels are also significant at beyond the 0.02 level (Chi-square >-5.41, df = 1). In general, once a given pair in either group began to fight, it usually fought on all of the subsequent tests.
When the female mice were 30 days old, all females were ovariectomized under ether anaesthesia and isolated in individual pans (4 × 10 × 5 in.). All mice were randomly divided into one of two groups: females in one group were subcutaneously administered I00 ~tg testosterone propionate (TP) each day for 20 days (the TP-Female group, n -- 32); a second group was administered a control injection of peanut oil each day for 20 days (Oil-Female group, n = 32). At the end of this treatment period, hormone injections were not given for 45 days to allow for the complete dissipation of any residual testosterone. At the end of this 45 day period all mice were tested in pairs for aggressive behavior, once a day for 2 successive days. Beginning on the day following the second test, all females were administered 10 ~tg TP each day for one week. On the 6th and 7th days of this week females were tested again for aggressive behavior. On the subsequent 4 weeks, all mice received daily injections of 20 ~tg, 50 Fg, 100 sg and 500 ~tg TP respectively, and were tested on the 6th and 7th days of each week for fighting. F o r testing, 2 individuals from the same group were paired in a neutral cage for 10 rain. If fighting occurred before the end of the 10 min period, the test was scored as positive, and terminated by replacing each mouse in its home cage. A fight was scored if one or both of the mice in the pair persistently attempted to bite the other. If no fighting occurred during the 10 rain test, the test was scored negative, and terminated by replacing each mouse in its home cage. The same individuals were paired for each set of weekly tests. The neutral cage was identical in construction to the home cages of the individually housed mice. All behavior tests were
DISCUSSION
The results of this experiment clearly indicate that chronic treatment of immature female mice with exogenous androgen facilitates the adult display of aggression in response to adult androgen treatment, even when a period as long as 45 days without hormone treatment intervenes between the initial treatment and the later treatment. This masculinization of immature female mice by chronic androgen treatment appears to be similar to the masculinization effects reported by
90
;~- TP pre)reot
/
%,
/
4~- Oil pretreat 60
Z-
tj
£
/
30 ¸
1 --
) I0
I, 20
Testosterone
dose,
1
I
1
50
I00
5OO
~g
FIG. 1. The per cent of the total number of adult ovariectomized pairs in each group fighting at each dose level of testosterone. At a given dose level, data points reflect the per cent of pairs in each group which fought on at least one of the two tests at that dose level. The abcissa shows the adult dose levels of TP employed.
POST-NEONATAL ANDROGEN AND AGGRESSION
467
Edwards [2, 3] who gave a single injection of testosterone to female mice on the day of birth. It is worth pointing out that in earlier experiments [2, 3] 90-95 percent of pairs of males, or females given TP on the day of birth, fight following androgen administration in adulthood. In this experiment, chronic TP administration begun at 30 days of age and continued for 20 days resulted in only 75 per cent of the pairs of pretreated females fighting following subsequent androgen treatment and testing in adulthood. This difference might indicate that a single injection of TP on the day of birth is relatively more effective in masculinizing the female with respect to later aggressive behavior than chronic androgen treatment begun as late as 30 days after birth. While this may be true, the present data make it clear that pretreatment of female mice with androgen between the age of 30 and 49 days facilitates the later display of androgen aroused aggressive behavior, and such facilitation appears qualitatively similar to that seen in females treated with TP on the day of birth. It might be argued that the enhanced aggressiveness in the adult females pretreated with androgen from 30-49 days of age might simply be due to the additive effects of residual hormones from the pretreatment with hormones administered in adulthood. According to this argument, enhanced aggressiveness in the TP-female group in adulthood would be due simply to the greater total amount of androgens available to the system during testing. The fact that a 45-day interval, during which no hormones were administered, separated pretreatment with subsequent treatment and testing speaks strongly against this possibility. Presuming that androgens arouse aggressiveness in mice
by acting on neural structures, these results indicate that pretreatment of immature females with chronic androgen stimulation effects some presumably permanent change in those neural mechanisms mediating the androgen control of aggression in mice. The exact nature of these changes, structural and/or biochemical remains to be determined. The ability of androgens administered to the neonate to permanently inhibit the ability of the organism to show lordosis in response to female sex hormones has been well documented for the rat [4, 6, 7] and the mouse (Edwards and Burge, unpublished observations). It has generally been found that the potential for androgen administered early in life to inhibit adult responsiveness to female sex hormones is restricted to some limited period of development: in the rat and mouse, during the first 10 days after birth. Although it is not yet clear whether the post-neonatal masculinization with respect to aggressive behavior in this experiment is a function of total hormone dose, or chronic administration of androgen for an extended period of time, or both, it is clear that the potential for masculinization of female mice with respect to testosterone aroused aggressive behavior exists for at least one month after birth. It might be expected that similar androgenic manipulation of female rats and mice would permanently inhibit adult responsiveness to female sex hormones. This hypothesis is probably not tenable, at least for the rat, since Wilson, Hamilton and Young [8] showed that treatment of female rats with androgen for 28 days does not inhibit the potential of the female to show lordosis when administered female sex hormones if androgen treatment is begun 15 days after birth. Whether similar results would obtain for the mouse remains to be determined.
REFERENCES
1. Beoman, E. A. The effect of male hormone on aggressive behavior in mice. Physiol. Zool. 20: 373--405, 1947. 2. Edwards, D. A. Mice: Fighting by neonatally androgenized females. Science 161: 1027-1028, 1968. 3. Edwards, D. A. Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behav. 4: 333-338, 1969. 4. Grady, K. L., C. H. Phoenix and W. C. Young. Role of the developing rat testis in differentiation of the neural tissue mediating mating behavior. J. comp. physiol. Psychol. 59: 176-182, 1965.
5. Tollman, J. and J. A. King. The effects of testosterone proprionate on aggression in male and female C57 B1/10 mice. Br. J. anita. Behav. 6: 147-149, 1956. 6. Whalen, R. E. and D. A. Edwards. Sexual reversability in neonatally castrated male rats. J. comp. physiol.Psychol. 57:175-182, 1966. 7. Whalen, R. E. and D. A. Edwards. Hormonal determinants of the development of masculine and feminine behavior in male and female rats. Anat. Rec. 157: 173-180, 1967. 8. Wilson, J. G., J. B. Hamilton and W. C. Young. Influence of age and presence of the ovaries on reproductive function in rats injected with androgens. Endocrinology 29: 784-789, 1941.
Post-Neonatal Androgenization and Adult Aggressive Behavior in Female Mice' D A V I D A. E D W A R D S
Department of Psychology, Emory University, Atlanta, Georgia, U.S.A. (Received 23 S e p t e m b e r 1969) EDWARDS, D. A. Post-neonatal androgenization and adult aggressive behavior in female mice. PHYSIOL. BEHAV. 5 (4) 465--467, 1970.--This experiment sought to determine whether chronic, long term administration of testosterone propionate (TP) to post-neonatal female mice would facilitate the androgen induced arousal of aggressive behavior in adulthood. Female mice were ovariectomized at 30 days of age and either administered TP daily for 20 days or a control injection of oil for 20 days. Forty-five days after the termination of the pretreatment, all mice were given TP and tested in pairs for fighting. Following androgen administration in adulthood 75 per cent of the pairs pretreated with TP in immaturity fought, but only 25 per cent of the pairs of females pretreated with oil in immaturity fought. It was concluded that the potential for androgen administered in infancy to induce masculinization of female mice with respect to androgen aroused aggressive behavior in adulthood exists for at least one month after birth. Mice
Androgen
Brain differentiation
Aggression
IT HAS BEEN known for some years that isolated male mice readily fight when paired. Castration of prepuberaily isolated male mice results in the failure of the mice to display aggressive behavior when a period of 25 or more days elapses between castration and testing: replacement therapy with exogenous androgen induces castrates to fight with other mice [1 ]. Thus, aggressive behavior in male mice is, at least partially, dependent upon androgen stimulation. In contrast to male mice, female mice rarely fight. Female mice do not even fight when administered large amounts of exogenous androgen [5]. Presuming that testosterone exerts its effects upon aggressive behavior through its action on central nervous system structures, these findings suggest that male and female mice possess functionally or structurally different neuroendocrine mechanisms relating to the control of aggression. In a recent series of experiments, Edwards [2, 3] showed that male mice and female mice given testosterone propionate (TP) on the day of birth displayed comparable levels of aggression when gonadectomized and tested for fighting following androgen administration in adulthood. Female mice treated only with oil at birth, failed to show aggression even when given large amounts of exogenous androgen in adulthood. Females given TP on the 10th day after birth showed more aggression than normal females, but less aggression than females given TP on the day of birth. These findings indicate that androgen stimulation of the neonatal female mouse masculinizes the female with respect to aggressiveness by causing or facilitating the differentiation of neuroendocrine mechanisms for aggression. Furthermore, the finding that females given TP 10 days after birth are less aggressive as
Development
adults than females given testosterone on the day of birth indicates the existence of a critical period for the androgen induced organization of mechanisms for aggression such that by the 10th day after birth the potential for such differentiation to occur is somewhat attenuated. While Edwards' findings indicate the existence of a critical period for the masculinization process in mice with respect to aggressive behavior, it should be recognized that the limits of the critical period are undoubtedly affected by the dose level of masculinizing androgen administered. Thus, Edwards' findings do not necessarily indicate that the potential for the androgen induced differentiation of hormone sensitive mechanisms for aggression is totally absent after the first few weeks after birth; rather, the results indicate that the capacity for a single injection of a given amount of androgen to facilitate aggressive behavior in the female is diminished by the 10th day after birth. It might be possible, for example, to masculinize female mice, with respect to aggressive behavior, much later in infancy or early adulthood if more extreme androgen manipulations were performed. This study reports the successful attempt to behaviorally masculinize female mice with respect to aggressive behavior by chronic androgen administration begun when the female mice were 30 days of age. METHOD
Subjects Sixty-four female Swiss-Webster mice (commercially obtained: Simonson Laboratories, Gilroy, California), 30
Wartial support was provided by a Pre-doctoral Research Fellowship from the USPHS (MH 30, 315-03), by grant HD 00893 from the National Institute of Child Health and Human Development to R. E. Whalen, by a research grant from Emory University, and by a Public Health Service Research Grant MH 17291 to D. A. Edwards. The testosterone used was generously supplied by Dr. Preston Perlman, Sebering Corporation, Bloomfield, New Jersey. 465
466
i!DWA
days old at the start of the experiment were raised and maintained under a reversed light-dark cycle with food and water ad lib.
t~.I ~F;
conducted during the first few hours of the dark phase of the lighting cycle. RESULTS
Procedure
As shown in Fig. 1, adult androgen administration lo animals in each of the experimental groups resulted in an increase in the proportion of pairs in each of the groups fighting. However, the effects of TP administration in adulthood upon fighting are much more striking for the group pretreated with testosterone in immaturity than for the group receiving control injections of oil during immaturity. In adulthood, 75 per cent of the pairs in the TP-female group fought at least once during the series of tests, but only 25 per cent of the pairs in the Oil-female group fought at least once. This difference is significant at beyond the 0.01 level (Chi-square = 8.0, df = 1). While very few pairs in either group fought at the lower hormone levels, a significantly greater proportion of TP-female pairs fought than Oil-female pairs at the 50 izg level. (Chi-square = 5.92, df = 1, p < 0.02). At the 100 izg and 500 t~g levels, TP-females fought more than Oil-females, and differences at each of these levels are also significant at beyond the 0.02 level (Chi-square >-5.41, df = 1). In general, once a given pair in either group began to fight, it usually fought on all of the subsequent tests.
When the female mice were 30 days old, all females were ovariectomized under ether anaesthesia and isolated in individual pans (4 × 10 × 5 in.). All mice were randomly divided into one of two groups: females in one group were subcutaneously administered I00 ~tg testosterone propionate (TP) each day for 20 days (the TP-Female group, n -- 32); a second group was administered a control injection of peanut oil each day for 20 days (Oil-Female group, n = 32). At the end of this treatment period, hormone injections were not given for 45 days to allow for the complete dissipation of any residual testosterone. At the end of this 45 day period all mice were tested in pairs for aggressive behavior, once a day for 2 successive days. Beginning on the day following the second test, all females were administered 10 ~tg TP each day for one week. On the 6th and 7th days of this week females were tested again for aggressive behavior. On the subsequent 4 weeks, all mice received daily injections of 20 ~tg, 50 Fg, 100 sg and 500 ~tg TP respectively, and were tested on the 6th and 7th days of each week for fighting. F o r testing, 2 individuals from the same group were paired in a neutral cage for 10 rain. If fighting occurred before the end of the 10 min period, the test was scored as positive, and terminated by replacing each mouse in its home cage. A fight was scored if one or both of the mice in the pair persistently attempted to bite the other. If no fighting occurred during the 10 rain test, the test was scored negative, and terminated by replacing each mouse in its home cage. The same individuals were paired for each set of weekly tests. The neutral cage was identical in construction to the home cages of the individually housed mice. All behavior tests were
DISCUSSION
The results of this experiment clearly indicate that chronic treatment of immature female mice with exogenous androgen facilitates the adult display of aggression in response to adult androgen treatment, even when a period as long as 45 days without hormone treatment intervenes between the initial treatment and the later treatment. This masculinization of immature female mice by chronic androgen treatment appears to be similar to the masculinization effects reported by
90
;~- TP pre)reot
/
%,
/
4~- Oil pretreat 60
Z-
tj
£
/
30 ¸
1 --
) I0
I, 20
Testosterone
dose,
1
I
1
50
I00
5OO
~g
FIG. 1. The per cent of the total number of adult ovariectomized pairs in each group fighting at each dose level of testosterone. At a given dose level, data points reflect the per cent of pairs in each group which fought on at least one of the two tests at that dose level. The abcissa shows the adult dose levels of TP employed.
POST-NEONATAL ANDROGEN AND AGGRESSION
467
Edwards [2, 3] who gave a single injection of testosterone to female mice on the day of birth. It is worth pointing out that in earlier experiments [2, 3] 90-95 percent of pairs of males, or females given TP on the day of birth, fight following androgen administration in adulthood. In this experiment, chronic TP administration begun at 30 days of age and continued for 20 days resulted in only 75 per cent of the pairs of pretreated females fighting following subsequent androgen treatment and testing in adulthood. This difference might indicate that a single injection of TP on the day of birth is relatively more effective in masculinizing the female with respect to later aggressive behavior than chronic androgen treatment begun as late as 30 days after birth. While this may be true, the present data make it clear that pretreatment of female mice with androgen between the age of 30 and 49 days facilitates the later display of androgen aroused aggressive behavior, and such facilitation appears qualitatively similar to that seen in females treated with TP on the day of birth. It might be argued that the enhanced aggressiveness in the adult females pretreated with androgen from 30-49 days of age might simply be due to the additive effects of residual hormones from the pretreatment with hormones administered in adulthood. According to this argument, enhanced aggressiveness in the TP-female group in adulthood would be due simply to the greater total amount of androgens available to the system during testing. The fact that a 45-day interval, during which no hormones were administered, separated pretreatment with subsequent treatment and testing speaks strongly against this possibility. Presuming that androgens arouse aggressiveness in mice
by acting on neural structures, these results indicate that pretreatment of immature females with chronic androgen stimulation effects some presumably permanent change in those neural mechanisms mediating the androgen control of aggression in mice. The exact nature of these changes, structural and/or biochemical remains to be determined. The ability of androgens administered to the neonate to permanently inhibit the ability of the organism to show lordosis in response to female sex hormones has been well documented for the rat [4, 6, 7] and the mouse (Edwards and Burge, unpublished observations). It has generally been found that the potential for androgen administered early in life to inhibit adult responsiveness to female sex hormones is restricted to some limited period of development: in the rat and mouse, during the first 10 days after birth. Although it is not yet clear whether the post-neonatal masculinization with respect to aggressive behavior in this experiment is a function of total hormone dose, or chronic administration of androgen for an extended period of time, or both, it is clear that the potential for masculinization of female mice with respect to testosterone aroused aggressive behavior exists for at least one month after birth. It might be expected that similar androgenic manipulation of female rats and mice would permanently inhibit adult responsiveness to female sex hormones. This hypothesis is probably not tenable, at least for the rat, since Wilson, Hamilton and Young [8] showed that treatment of female rats with androgen for 28 days does not inhibit the potential of the female to show lordosis when administered female sex hormones if androgen treatment is begun 15 days after birth. Whether similar results would obtain for the mouse remains to be determined.
REFERENCES
1. Beoman, E. A. The effect of male hormone on aggressive behavior in mice. Physiol. Zool. 20: 373--405, 1947. 2. Edwards, D. A. Mice: Fighting by neonatally androgenized females. Science 161: 1027-1028, 1968. 3. Edwards, D. A. Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behav. 4: 333-338, 1969. 4. Grady, K. L., C. H. Phoenix and W. C. Young. Role of the developing rat testis in differentiation of the neural tissue mediating mating behavior. J. comp. physiol. Psychol. 59: 176-182, 1965.
5. Tollman, J. and J. A. King. The effects of testosterone proprionate on aggression in male and female C57 B1/10 mice. Br. J. anita. Behav. 6: 147-149, 1956. 6. Whalen, R. E. and D. A. Edwards. Sexual reversability in neonatally castrated male rats. J. comp. physiol.Psychol. 57:175-182, 1966. 7. Whalen, R. E. and D. A. Edwards. Hormonal determinants of the development of masculine and feminine behavior in male and female rats. Anat. Rec. 157: 173-180, 1967. 8. Wilson, J. G., J. B. Hamilton and W. C. Young. Influence of age and presence of the ovaries on reproductive function in rats injected with androgens. Endocrinology 29: 784-789, 1941.