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The effect of the antiestrogen CI-628 on androgen-induced aggressive behavior in castrated male mice
HORMONES
AND
BEHAVIOR
12, 205-210 (1979)
The Effect of the Antiestrogen Cl-628 on AndrogenInduced Aggressive Behavior in Castrated Male Mice C. R. CLARK AND N. W. NOWELL Department
of Zoology,
University
of Hull, Hull, North Humberside,
HU4 7RX, England
This study was conducted to determine whether or not the antiestrogen U-628 would block testosterone-maintained fighting in castrated male mice. TO strain adult male mice were castrated and injected S.C.every day for 14 days with either (1) 75 pg testosterone or (2) 75 pg testosterone and 1 mg U-628, and in addition 1 mg of U-628 6 hr prior to each injection of antiestrogen and androgen. Vehicleinjected, castrated, and CI-628-injected animals were employed as controls. Testosterone-maintained intermale aggressive behavior was blocked by the antiestrogen CI-628. This study provides support for the hypothesis that testosterone exerts its effects on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites.
The importance of testosterone in the expression of intermale aggressive behavior in the mammal is well established (Edwards and Rowe, 1975). Evidence is accumulating that the central action of testosterone may involve its intraneuronal aromatization to estrogen (Naftolin, Ryan, Davies, Reddy, Flores, Petro, and Kuhn, 1975). In this regard several studies have demonstrated the capacity of the mammalian brain to metabolize androgen to estrogen both in viva (Lieberburg and McEwen, 1975) and in vitro (Naftolin, Ryan, and Petro, 1971). Nitromophene citrate (CI-628) is a potent nonsteroidal antiestrogen in both peripheral and central nervous target tissues for estradiol. For example, Chazal, Faudon, Gogan, and Rotsztein (1975) demonstrated that significant (X-628 inhibition of [3H]estradiol binding occurs in the central nervous system as measured in whole homogenates and nuclear fractions. Furthermore, Roy and Wade (1977) have reported that CI-628 also has the ability to displace estradiol which has previously bound to specific neural receptors. This was evidenced when CI-628 was injected 2 hr prior to an injection of [3H]estradiol. This resulted in a greater inhibition of nuclear estradiol retention at 12 hr after the [3H]estradiol injection than at 2 hr. These studies illustrate the potent antiestrogenic activity of this compound within the central nervous system. 20.5 0018-506X/79/030205-06$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
206
CLARK
AND NOWELL
This present study attempted to show whether the antiestrogen U-628 would block testosterone-maintained fighting in castrated male mice. MATERIALS
AND METHODS
Breeding and housing regime of animals. The male albino TO strain mice used in this study were originally obtained from A. Tuck and Sons Ltd., Rayleigh, Essex, England. They were kept in conditions of minimum stress with a light regimen of 12 hr (0800-2000). The animal room temperature was maintained at 18-21°C with food and water supplied ad libitum. Litters were culled to six on day of birth and then remained unhandled until weaning at 18-22 days of age, when they were segregated into single-sex groups of six. Mice were 110 day old at the time of decapitation. Treatment. Fifty of these mice (71 day old) were caged singly for 3 weeks, orchidectomized via the abdominal route under ether anesthesia, and returned to their home cages for a period of 4 days. On the 5th day they were randomly divided into five equal groups: (1) injected subcutaneously (s.c.) every day for 14 days, with 75 pg of testosterone (free alcohol; Sigma Chemicals, Ltd.) dissolved in 200 ~1 of benzyl benzoate:arachis oil (1:20); (2) injected s.c, every day for 14 days with 75 pg of testosterone and 1 mg CI-628 (Parke-Davis, Pontypool, England), and in addition 1 mg of CI-628 6 hr prior to each injection of antiestrogen and androgen; (3) injected S.C. twice a day for 14 days with 200 ~1 of benzyl benzoate:arachis oil (1:20); (4) injected S.C.twice a day for 14 days with 2 mg of CI-628; (5) untreated. The timing of antihormone treatment in suppressing steroid-receptor binding is crucial (Clark and Nowell, 1979a). It was for this reason that CI-628 was given 6 hr prior to the combined injection of androgen and antiestrogen. Injections were given at different body sites and no evidence of body sores was found. Neither was the body weight affected by U-628 injections (41.2 g compared with 41.8 g for the oil-injected castrates). Testing procedure. Aggression tests were conducted during the last 3 days of androgen/antihormone treatment. The injections were timed so that treatment was as remote as possible from the aggression tests. Experimental mice were fought in their home cages against anosmic-grouped male standard opponents of similar weight and age. These latter stimulus males were rendered anosmic by intranasal application of 5% zinc sulfate solution (Alberts and Andrews, 1971). Anosmia was confirmed by the elimination of the avoidance response normally shown by male mice towards an area treated with urine of isolated male conspecifics (Jones and Nowell, 1976). Anosmic standard opponents were employed so as to eliminate any variation in their stimulus properties, which may be induced by a change in the pheromonal output of test animals injected with antihormone (Nowell and Wouters, 1972).
U-628 AND AGGRESSIVE BEHAVIOR
207
On each of the 3 consecutive days of aggression testing, stimulus males were transferred to the home cage of the test animal on a wire support. The latency in seconds to the first attack, the accumulated attack time and number of bites was recorded during a 5-min test period, after which the stimulus male was returned to its home cage. Immediately after the last bout, the experimental mice were decapitated and the weights of the ventral prostrate glands recorded. Statistics. Behavioral data were statistically analyzed (Campbell, 1974) employing the Mann-Whitney test. Mean relative organ weight data were contrasted between different treatment categories using the Student’s t test. RESULTS Aggression test results are presented in Table 1. Mice receiving testosterone showed significantly higher levels of aggression in all measures recorded, i.e., latency to attack (L.A.), accumulated attack time (A.A.T.), and number of bites (N.B.), P < 0.001, than mice receiving testosterone in combination with CI-628. There was no statistical difference in the L.A., A.A.T., or N.B. (P > 0.05) between untreated and CI-628-plus-testosterone-treated animals. Individuals receiving vehicle showed the same level of aggressive behavior as untreated controls (in all measures P > 0.05). CI-628 had no effect on the ventral prostrate, as indicated by weights of this organ in testosterone- and CI-628-plus-testosterone-treated mice (P > 0.05). CI-628 also had no effect on the weight of this organ when administered alone (P > 0.05), when compared with untreated mice. DISCUSSION This study has demonstrated that the antiestrogen U-628 has the capacity to inhibit aggressive behavior in testosterone-treated male castrated TABLE 1 The Effect of the Antiestrogen CI-628 on Aggressive Behavior and Ventral Prostate Weight of Male Mice Treatment
Testosterone Testosterone + CI-628 (X-628 Vehicle Untreated
Percentage pairs fighting
Latency to first attack bed
80
105.4 2 21.7
20 20 20 20
251.0 5 259.7 2 262.1 t 261.9 2
18.4 15.5 15.0 15.2
Accumulated attack time (set)
Relative ventral prostate weight
32.2 _c 4.2
43.5 t 1.9
3.4 5.2 4.3 3.8
-t t 2 t
1.4 1.8 1.7 1.7
(md100gm)
44.3 2 16.2 t 16.4 -t 15.7 f
2.2 1.9 1.5 1.1
208
CLARK
AND NOWELL
mice. Our results with the use of this estrogen-receptor antagonist agree with those of Bowden and Brain (1978) who found that the aromatization inhibitor 4-androstene-3,6,17-trione could block testosterone maintained fighting in castrated male mice. The results of the present study contrasts with the effects of the antiandrogens cyproterone acetate (Edwards, 1970; Clark and Nowell, 1979a) and flutamide (Clark and Nowell, 1979b) on aggressive behavior, where no effect of these antihormones was recorded. They do however agree with the predictions of the aromatization hypothesis (Plapinger and McEwen, 1978)that antiestrogens should block androgen-dependent behavior by competing with estrogen for specific neural receptors, whereas antiandrogens would have no effect. The suppression of intermale aggressive behavior by CI-628 is interpreted to represent an antiestrogenic rather than a nonspecific effect of this antihormone. This is suggested by the absence of body weight loss, as was found by Luttge, Hall, Wallis, and Campbell (1975), and the lack of extraneous sex steroid action as evidenced by ventral prostate weights (see also Callantine, Humphrey, Lee, Windsor, Schottin, and O’Brien, 1966). Failure of CI-628 to counteract androgen induced masculine sexual behavior in the rat has been reported (Whalen, Battie, and Luttge, 1972). Here rats were castrated and injected daily with CI-628 (1 mg) concurrently with testosterone for 8 weeks. However, in a more recent study, Luttge (1975) increased the dose to 2.5 mg CI-628 per injection and gave this antiestrogen 6 hr prior to and also concurrently with testosterone. Here CI-628 was found to reduce testosterone-stimulated male sexual behavior. In this present study we have employed a similar multiple injection schedule for CI-628, since we have previously demonstrated the importance of the timing of antihormone treatment in suppressing neural receptor binding of gonadal steroids (Clark and Nowell, 1979a). In agreement with the effect of CI-628 on male sexual behavior, Beyer, Morali, Naftolin, Larsson, and Perez-Palacois (1976), demonstrated that the antiestrogen (ICI 46474), and an aromatase inhibitor (aminoglutethimide) suppressed testosterone-induced mounting behavior in sexually inexperienced male rats. However, pretreatments with two other antiestrogens, i.e., MER-25 and clomiphene and two aromatase inhibitors, i.e., metopirone and Sa-androstenedione, did not inhibit the responses to testosterone. These authors (Beyer et al., 1976) concluded that in this case the dosages and schedules of treatment selected were inadequate to counteract the possible effects of estrogen on the mechanism related to the expression of male sexual behavior. The antiestrogenic property of CI-628 is due to its ability to compete with estradiol for specific neural receptors (Roy and Wade, 1977). One hypothesis to explain the inhibitory action of CI-628 states that the antiestrogen receptor complex, which is translocated into the nucleus, inhibits
Cl-628 AND AGGRESSIVE BEHAVIOR
209
the replenishment of estrogen cytosol receptors. More recently however, based on observations indicating a slow metabolic clearance of various nonsteroidal antiestrogens, including CI-628, Jordan, Dix, Naylor, Prestwith, and Rowsby (1978), argue that estrogen receptor resynthesis does occur after antiestrogen administration, but the newly synthesized receptors are never allowed to accumulate within the cytoplasm since antiestrogens from the blood immediately provoke translocation into the nucleus. The present study provides support for the hypothesis that testosterone exerts its effect on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites. ACKNOWLEDGMENTS The authors would like to thank Professor J. G. Phillips for facilities in the Department of Zoology and the Science Research Council for a Studentship awarded to C. R. Clark. We would also like to thank Dr. Gorringe, Parke-Davis Corporation, Pontypool, England, for the generous gift of (X-628.
REFERENCES Alberts, J. R., and Andrews, B. G., Jr. (1971). Acute anosmia in the rat: A behavioral test of a peripherally-induced olfactory deficit. Physiol. Behav. 6, 619-621. Beyer, C., Morali, G., Naftolin, F., Larsson, K., and Perez, G. (1976). Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats. Harm. Behav. 7, 353-363. Bowden, N. J., and Brain, P. F. (1978). Blockade of testosterone-maintained intermale fighting in albino laboratory mice by an aromatization inhibitor. Physiol. Behav. 20, 543-546. Callantine, M. R., Humphrey, R. R., Lea, S. L., Windsor, B. L., Schottin, N. H., and O’Brien, 0. P. (1966). Action of an oestrogen antagonist on reproductive mechanisms in the rat. Endocrinology 79, 153-167. Campbell, R. C. (1974). Stafisfics for Biologisrs. Cambridge Univ. Press, Cambridge. Chazal, G., Faudon, M., Gogan, F., and Rotsztein, W. (1975). Effect of two estradiol antagonists upon estradiol uptake in the rat brain and peripheral tissues. Bruin Res. 89, 245-254. Clark, C. R., and Nowell, N. W. (1979a). The effect of the antiandrogen cyproterone acetate on neural testosterone binding and aggressive behavior in the male mouse. J. Endocrinol. 81, 137-138. Clark, C. R., and Nowell, N. W. (1979b). The effect of the antiandrogen flutamide on neural receptor binding of testosterone and aggressive behavior in the male mouse. In press. Clark, J. H., Peck, E. J., Jr., and Anderson, J. N. (1974). Oestrogen receptors and antagonism of steroid hormone action. Nature (London) 251, 446-448. Edwards, D. A. (1970). Effects of cyproterone acetate on aggressive behavior and the seminal vesicles of male mice. J. Endocrinol. 46, 77-81. 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 Behaviour, pp. 273-303. Plenum Press, New York. Jones, R. B., and Nowell, N. W. (1976). Zinc sulphate anosmia eliminates the avoidance response to male mouse urine. Chem. Sen. Flav. 3, 129-133.
210
CLARK
AND
NOWELL
Jordan, V. C., Dix, C. J., Naylor, K. E., Prestwich, G., and Rowsby, L. (1978). Nonsteroida1antiestrogens: Their biological effects and potential mechanism of action. J. Toxicol. Environ.
Health
4, 363-390.
Lieberburg, I., and McEwen, B. S. (l975). Estradiol-17P: A metabolite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains. Brain Res. 91, 171-174. Luttge, W. G. (1975). Effects of antiestrogens on testosterone stimulated sexual behavior and peripheral target tissues in the castrate male rat. Physiol. Behav. 14, 839-846. Luttge, W. G., Hall, N. R., Wallis, C. J., and Campbell, J. C. (1975). Stimulation of male and female sexual behavior in gonadectomized rats with estrogen and androgen therapy and its inhibition with concurrent anti-hormone therapy. Physiol. Behav. 14, 65-73. Naftohn, F., Ryan, K. J., Petro, Z. (1971). Aromatization of androstenedione by the diencephalon. .I. C/in. Endocrinol. Metabol. 33, 368-370. Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., and Kuhn, M. (1975). The formation of estrogens by central neuroendocrine tissues. Rec. Prog. Horm. Res. 31, 295-319.
Nowell, N. W., and Wouters, A. (1972). The effect of cyproterone acetate upon aggressive behavior in the laboratory mouse. J. Endocrinol. 57, xxxvi-xxxvii. Plapinger, L., and McEwen, B. S. (1978). Gonadal steroid-brain interactions in sexual differentiation. In J. B. Hutchison (Eds.), Bio/ogica/ Determinants ofSexual Behavior, pp. 153-218. Wiley, New York. Roy, E., and Wade, G. N. (1977). Binding of 13H]-estradiol by brain cell nuclei and female rat sexual behavior: Inhibition by antiestrogens. Brain Res. 126, 73-87. Whalen, R. E., Battie, C., and Luttge, W. G. (1972). Antiestrogen inhibition of androgen induced sexual receptivity in rats. Behav. Biol. 7, 311-320.
AND
BEHAVIOR
12, 205-210 (1979)
The Effect of the Antiestrogen Cl-628 on AndrogenInduced Aggressive Behavior in Castrated Male Mice C. R. CLARK AND N. W. NOWELL Department
of Zoology,
University
of Hull, Hull, North Humberside,
HU4 7RX, England
This study was conducted to determine whether or not the antiestrogen U-628 would block testosterone-maintained fighting in castrated male mice. TO strain adult male mice were castrated and injected S.C.every day for 14 days with either (1) 75 pg testosterone or (2) 75 pg testosterone and 1 mg U-628, and in addition 1 mg of U-628 6 hr prior to each injection of antiestrogen and androgen. Vehicleinjected, castrated, and CI-628-injected animals were employed as controls. Testosterone-maintained intermale aggressive behavior was blocked by the antiestrogen CI-628. This study provides support for the hypothesis that testosterone exerts its effects on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites.
The importance of testosterone in the expression of intermale aggressive behavior in the mammal is well established (Edwards and Rowe, 1975). Evidence is accumulating that the central action of testosterone may involve its intraneuronal aromatization to estrogen (Naftolin, Ryan, Davies, Reddy, Flores, Petro, and Kuhn, 1975). In this regard several studies have demonstrated the capacity of the mammalian brain to metabolize androgen to estrogen both in viva (Lieberburg and McEwen, 1975) and in vitro (Naftolin, Ryan, and Petro, 1971). Nitromophene citrate (CI-628) is a potent nonsteroidal antiestrogen in both peripheral and central nervous target tissues for estradiol. For example, Chazal, Faudon, Gogan, and Rotsztein (1975) demonstrated that significant (X-628 inhibition of [3H]estradiol binding occurs in the central nervous system as measured in whole homogenates and nuclear fractions. Furthermore, Roy and Wade (1977) have reported that CI-628 also has the ability to displace estradiol which has previously bound to specific neural receptors. This was evidenced when CI-628 was injected 2 hr prior to an injection of [3H]estradiol. This resulted in a greater inhibition of nuclear estradiol retention at 12 hr after the [3H]estradiol injection than at 2 hr. These studies illustrate the potent antiestrogenic activity of this compound within the central nervous system. 20.5 0018-506X/79/030205-06$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
206
CLARK
AND NOWELL
This present study attempted to show whether the antiestrogen U-628 would block testosterone-maintained fighting in castrated male mice. MATERIALS
AND METHODS
Breeding and housing regime of animals. The male albino TO strain mice used in this study were originally obtained from A. Tuck and Sons Ltd., Rayleigh, Essex, England. They were kept in conditions of minimum stress with a light regimen of 12 hr (0800-2000). The animal room temperature was maintained at 18-21°C with food and water supplied ad libitum. Litters were culled to six on day of birth and then remained unhandled until weaning at 18-22 days of age, when they were segregated into single-sex groups of six. Mice were 110 day old at the time of decapitation. Treatment. Fifty of these mice (71 day old) were caged singly for 3 weeks, orchidectomized via the abdominal route under ether anesthesia, and returned to their home cages for a period of 4 days. On the 5th day they were randomly divided into five equal groups: (1) injected subcutaneously (s.c.) every day for 14 days, with 75 pg of testosterone (free alcohol; Sigma Chemicals, Ltd.) dissolved in 200 ~1 of benzyl benzoate:arachis oil (1:20); (2) injected s.c, every day for 14 days with 75 pg of testosterone and 1 mg CI-628 (Parke-Davis, Pontypool, England), and in addition 1 mg of CI-628 6 hr prior to each injection of antiestrogen and androgen; (3) injected S.C. twice a day for 14 days with 200 ~1 of benzyl benzoate:arachis oil (1:20); (4) injected S.C.twice a day for 14 days with 2 mg of CI-628; (5) untreated. The timing of antihormone treatment in suppressing steroid-receptor binding is crucial (Clark and Nowell, 1979a). It was for this reason that CI-628 was given 6 hr prior to the combined injection of androgen and antiestrogen. Injections were given at different body sites and no evidence of body sores was found. Neither was the body weight affected by U-628 injections (41.2 g compared with 41.8 g for the oil-injected castrates). Testing procedure. Aggression tests were conducted during the last 3 days of androgen/antihormone treatment. The injections were timed so that treatment was as remote as possible from the aggression tests. Experimental mice were fought in their home cages against anosmic-grouped male standard opponents of similar weight and age. These latter stimulus males were rendered anosmic by intranasal application of 5% zinc sulfate solution (Alberts and Andrews, 1971). Anosmia was confirmed by the elimination of the avoidance response normally shown by male mice towards an area treated with urine of isolated male conspecifics (Jones and Nowell, 1976). Anosmic standard opponents were employed so as to eliminate any variation in their stimulus properties, which may be induced by a change in the pheromonal output of test animals injected with antihormone (Nowell and Wouters, 1972).
U-628 AND AGGRESSIVE BEHAVIOR
207
On each of the 3 consecutive days of aggression testing, stimulus males were transferred to the home cage of the test animal on a wire support. The latency in seconds to the first attack, the accumulated attack time and number of bites was recorded during a 5-min test period, after which the stimulus male was returned to its home cage. Immediately after the last bout, the experimental mice were decapitated and the weights of the ventral prostrate glands recorded. Statistics. Behavioral data were statistically analyzed (Campbell, 1974) employing the Mann-Whitney test. Mean relative organ weight data were contrasted between different treatment categories using the Student’s t test. RESULTS Aggression test results are presented in Table 1. Mice receiving testosterone showed significantly higher levels of aggression in all measures recorded, i.e., latency to attack (L.A.), accumulated attack time (A.A.T.), and number of bites (N.B.), P < 0.001, than mice receiving testosterone in combination with CI-628. There was no statistical difference in the L.A., A.A.T., or N.B. (P > 0.05) between untreated and CI-628-plus-testosterone-treated animals. Individuals receiving vehicle showed the same level of aggressive behavior as untreated controls (in all measures P > 0.05). CI-628 had no effect on the ventral prostrate, as indicated by weights of this organ in testosterone- and CI-628-plus-testosterone-treated mice (P > 0.05). CI-628 also had no effect on the weight of this organ when administered alone (P > 0.05), when compared with untreated mice. DISCUSSION This study has demonstrated that the antiestrogen U-628 has the capacity to inhibit aggressive behavior in testosterone-treated male castrated TABLE 1 The Effect of the Antiestrogen CI-628 on Aggressive Behavior and Ventral Prostate Weight of Male Mice Treatment
Testosterone Testosterone + CI-628 (X-628 Vehicle Untreated
Percentage pairs fighting
Latency to first attack bed
80
105.4 2 21.7
20 20 20 20
251.0 5 259.7 2 262.1 t 261.9 2
18.4 15.5 15.0 15.2
Accumulated attack time (set)
Relative ventral prostate weight
32.2 _c 4.2
43.5 t 1.9
3.4 5.2 4.3 3.8
-t t 2 t
1.4 1.8 1.7 1.7
(md100gm)
44.3 2 16.2 t 16.4 -t 15.7 f
2.2 1.9 1.5 1.1
208
CLARK
AND NOWELL
mice. Our results with the use of this estrogen-receptor antagonist agree with those of Bowden and Brain (1978) who found that the aromatization inhibitor 4-androstene-3,6,17-trione could block testosterone maintained fighting in castrated male mice. The results of the present study contrasts with the effects of the antiandrogens cyproterone acetate (Edwards, 1970; Clark and Nowell, 1979a) and flutamide (Clark and Nowell, 1979b) on aggressive behavior, where no effect of these antihormones was recorded. They do however agree with the predictions of the aromatization hypothesis (Plapinger and McEwen, 1978)that antiestrogens should block androgen-dependent behavior by competing with estrogen for specific neural receptors, whereas antiandrogens would have no effect. The suppression of intermale aggressive behavior by CI-628 is interpreted to represent an antiestrogenic rather than a nonspecific effect of this antihormone. This is suggested by the absence of body weight loss, as was found by Luttge, Hall, Wallis, and Campbell (1975), and the lack of extraneous sex steroid action as evidenced by ventral prostate weights (see also Callantine, Humphrey, Lee, Windsor, Schottin, and O’Brien, 1966). Failure of CI-628 to counteract androgen induced masculine sexual behavior in the rat has been reported (Whalen, Battie, and Luttge, 1972). Here rats were castrated and injected daily with CI-628 (1 mg) concurrently with testosterone for 8 weeks. However, in a more recent study, Luttge (1975) increased the dose to 2.5 mg CI-628 per injection and gave this antiestrogen 6 hr prior to and also concurrently with testosterone. Here CI-628 was found to reduce testosterone-stimulated male sexual behavior. In this present study we have employed a similar multiple injection schedule for CI-628, since we have previously demonstrated the importance of the timing of antihormone treatment in suppressing neural receptor binding of gonadal steroids (Clark and Nowell, 1979a). In agreement with the effect of CI-628 on male sexual behavior, Beyer, Morali, Naftolin, Larsson, and Perez-Palacois (1976), demonstrated that the antiestrogen (ICI 46474), and an aromatase inhibitor (aminoglutethimide) suppressed testosterone-induced mounting behavior in sexually inexperienced male rats. However, pretreatments with two other antiestrogens, i.e., MER-25 and clomiphene and two aromatase inhibitors, i.e., metopirone and Sa-androstenedione, did not inhibit the responses to testosterone. These authors (Beyer et al., 1976) concluded that in this case the dosages and schedules of treatment selected were inadequate to counteract the possible effects of estrogen on the mechanism related to the expression of male sexual behavior. The antiestrogenic property of CI-628 is due to its ability to compete with estradiol for specific neural receptors (Roy and Wade, 1977). One hypothesis to explain the inhibitory action of CI-628 states that the antiestrogen receptor complex, which is translocated into the nucleus, inhibits
Cl-628 AND AGGRESSIVE BEHAVIOR
209
the replenishment of estrogen cytosol receptors. More recently however, based on observations indicating a slow metabolic clearance of various nonsteroidal antiestrogens, including CI-628, Jordan, Dix, Naylor, Prestwith, and Rowsby (1978), argue that estrogen receptor resynthesis does occur after antiestrogen administration, but the newly synthesized receptors are never allowed to accumulate within the cytoplasm since antiestrogens from the blood immediately provoke translocation into the nucleus. The present study provides support for the hypothesis that testosterone exerts its effect on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites. ACKNOWLEDGMENTS The authors would like to thank Professor J. G. Phillips for facilities in the Department of Zoology and the Science Research Council for a Studentship awarded to C. R. Clark. We would also like to thank Dr. Gorringe, Parke-Davis Corporation, Pontypool, England, for the generous gift of (X-628.
REFERENCES Alberts, J. R., and Andrews, B. G., Jr. (1971). Acute anosmia in the rat: A behavioral test of a peripherally-induced olfactory deficit. Physiol. Behav. 6, 619-621. Beyer, C., Morali, G., Naftolin, F., Larsson, K., and Perez, G. (1976). Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats. Harm. Behav. 7, 353-363. Bowden, N. J., and Brain, P. F. (1978). Blockade of testosterone-maintained intermale fighting in albino laboratory mice by an aromatization inhibitor. Physiol. Behav. 20, 543-546. Callantine, M. R., Humphrey, R. R., Lea, S. L., Windsor, B. L., Schottin, N. H., and O’Brien, 0. P. (1966). Action of an oestrogen antagonist on reproductive mechanisms in the rat. Endocrinology 79, 153-167. Campbell, R. C. (1974). Stafisfics for Biologisrs. Cambridge Univ. Press, Cambridge. Chazal, G., Faudon, M., Gogan, F., and Rotsztein, W. (1975). Effect of two estradiol antagonists upon estradiol uptake in the rat brain and peripheral tissues. Bruin Res. 89, 245-254. Clark, C. R., and Nowell, N. W. (1979a). The effect of the antiandrogen cyproterone acetate on neural testosterone binding and aggressive behavior in the male mouse. J. Endocrinol. 81, 137-138. Clark, C. R., and Nowell, N. W. (1979b). The effect of the antiandrogen flutamide on neural receptor binding of testosterone and aggressive behavior in the male mouse. In press. Clark, J. H., Peck, E. J., Jr., and Anderson, J. N. (1974). Oestrogen receptors and antagonism of steroid hormone action. Nature (London) 251, 446-448. Edwards, D. A. (1970). Effects of cyproterone acetate on aggressive behavior and the seminal vesicles of male mice. J. Endocrinol. 46, 77-81. 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 Behaviour, pp. 273-303. Plenum Press, New York. Jones, R. B., and Nowell, N. W. (1976). Zinc sulphate anosmia eliminates the avoidance response to male mouse urine. Chem. Sen. Flav. 3, 129-133.
210
CLARK
AND
NOWELL
Jordan, V. C., Dix, C. J., Naylor, K. E., Prestwich, G., and Rowsby, L. (1978). Nonsteroida1antiestrogens: Their biological effects and potential mechanism of action. J. Toxicol. Environ.
Health
4, 363-390.
Lieberburg, I., and McEwen, B. S. (l975). Estradiol-17P: A metabolite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains. Brain Res. 91, 171-174. Luttge, W. G. (1975). Effects of antiestrogens on testosterone stimulated sexual behavior and peripheral target tissues in the castrate male rat. Physiol. Behav. 14, 839-846. Luttge, W. G., Hall, N. R., Wallis, C. J., and Campbell, J. C. (1975). Stimulation of male and female sexual behavior in gonadectomized rats with estrogen and androgen therapy and its inhibition with concurrent anti-hormone therapy. Physiol. Behav. 14, 65-73. Naftohn, F., Ryan, K. J., Petro, Z. (1971). Aromatization of androstenedione by the diencephalon. .I. C/in. Endocrinol. Metabol. 33, 368-370. Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., and Kuhn, M. (1975). The formation of estrogens by central neuroendocrine tissues. Rec. Prog. Horm. Res. 31, 295-319.
Nowell, N. W., and Wouters, A. (1972). The effect of cyproterone acetate upon aggressive behavior in the laboratory mouse. J. Endocrinol. 57, xxxvi-xxxvii. Plapinger, L., and McEwen, B. S. (1978). Gonadal steroid-brain interactions in sexual differentiation. In J. B. Hutchison (Eds.), Bio/ogica/ Determinants ofSexual Behavior, pp. 153-218. Wiley, New York. Roy, E., and Wade, G. N. (1977). Binding of 13H]-estradiol by brain cell nuclei and female rat sexual behavior: Inhibition by antiestrogens. Brain Res. 126, 73-87. Whalen, R. E., Battie, C., and Luttge, W. G. (1972). Antiestrogen inhibition of androgen induced sexual receptivity in rats. Behav. Biol. 7, 311-320.