Testosterone propionate inhibits maternal aggression in mice

Physiology & Behavior, Vol. 24, pp. 435--439. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.

Testosterone Propionate Inhibits Maternal Aggression in Mice BRUCE SVARE

Department of Psychology, State University of New York at Albany 1400 Washington Avenue, Albany, N Y 12222 Received 21 August 1979 SVARE, B. Testosterone propionate inhibits maternal aggression in mice. PHYSIOL. BEHAV. 24(3) 435--439, 1980.--Dally injections of testosterone propionate (TP) significantly decreased the number of attacks exhibited by lactating female mice toward male intruders. Treatment with TP also depressed the body weights of the dams as well as their lactation performance but these effects were observed long after deficits in aggression were noted. The results are discussed in terms of a direct effect of the steroid on central neural tissue mediating the behavior.

Mice

Lactation

Maternalaggression

Testosterone propionate

WHEN threatened by strange conspecifics, lactating females of most mammalian species exhibit intense aggressive behavior, referred to here as "maternal aggression". While a number of biological and environmental events are known to influence the initiation and maintenance of the response in rodents, the exact nature of the biochemical events controlling the behavior still remain to be elucidated (for recent reviews see [17,18]). It is well-known that the primary male androgen, testosterone (T), can suppress maternal behavior and promote aggressive behavior. In the case of the latter, exposure to T early in life is known to sensitize neural tissue and behavior in a male direction such that intermale aggression will be promoted following adult exposure to the steroid [3, 24, 26]. More recent studies indicate that adult T may modulate female aggressive behavior as well. While most mammalian females are aggressive only during lactation, hamsters and Peromyscus mice are also aggressive toward conspecifics during the virgin state. Ovariectomy reduces the behavior while testosterone propionate (TP) replacement therapy restores it [9,10]. Testosterone also is known to suppress a number of different aspects of maternal behavior. In the virgin female rat and mouse, TP inhibits pup retrieval, anogenital licking, and nursing while promoting pup-killing behavior [5,14]. Also, in the mouse, the administration of TP during gestation suppresses maternal nest building [7] while postpartum TP treatment inhibits lactation [8]. Will TP treatment adversely affect another aspect of maternal behvior, aggression, or will it promote the behavior in a manner similar to other agonistic responses? The following experiment was designed to examine the above question by assessing the effects of TP on maternal aggression in lactating mice.

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METHOD

Animals Pregnant multiparous Rockland-Swiss (R-S) albino mice were housed individually in l l x 7 x 5 in. translucent cages with wood chips on the floor. The mice had free access to food (Charles River Mouse Chow) and water and were mainrained on a 12/12 hr light/dark cycle with lights on between 7 a.m. and 7 p.m. Testing was conducted at 10 a.m. Procedure

Screening. Screening of animals for maternal aggression was necessary because about 30% of R-S mice do not exhibit the behavior. On the 2nd postpartum day (litters were reduced to 6 pups on the day of parturition) a group housed (6/cage) adult male R-S mouse was introduced into the homecage of each dam 3 min following the removal of its young. The young were removed to prevent them from interfeting with the behavioral interactions. This period of pup removal was used on subsequent tests and has been shown in previous work not to influence maternal aggression [6,20]. If the dam bit and chased the intruder during the 3 min test, it was scored -as exhibiting fighting. The intruder animals were removed as soon as a fight was observed and were used for only one aggression test. Group housed intruder males are good stimuli for maternal aggression testing because they rarely attack lactating animals or fight back in response to being attacked. Surgery, treatments, and testing. Thirty-two lactating animals that exhibited maternal aggression, were bilaterally ovariectomized under ether anesthesia. Ovariectomy was performed in order to prevent possible contamination by ovarian steroids. Numerous studies indicate that the loss of

~Address reprint requests to: Dr. Bruce B. Svare, Department of Psychology, State University of New York at Albany, 1400 Washington Avenue, Albany, NY 12222.

Copyright © 1980 Brain Research Publications Inc.m0031-9384/80/030435-05802.00/0

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FIG. 1. The number of attacks exhibited toward a male intruder by lactating female R-S mice treated daily from Postpartum Days 4-13 with oil, 125, 250, or 500 txg testosterone propionate (TP). Behavioral tests were conducted at 2 day intervals on Days 4 (prior to the first injection), 6, 8, 10, 12, and 14 of the lactation period.

ovarian hormones does not alter postpartum maternal and aggressive behaviors in the mouse [23]. The animals were randomly divided into equal groups (n=8/group) consisting of the following treatment conditions: (1) 125 f g testosterone propionate (TP), (2) 250 fig TP, (3) 500 f g TP, and (4) oil. The above range of TP doses was selected on the basis of their demonstrated effectiveness in promoting aggressive behavior in male and virgin female mice and inhibiting maternal behaviors in pregnant and lactating female mice [7, 9, 10]. The hormones were dissolved in 0.1 cc sesame oil and administered subcutaneously once a day at 11:00 a.m. for 10 days beginning on the 4th postpartum day. The oil control group was given an equal volume of the vehicle. Three minute aggression tests against a male intruder were conducted at 2 day intervals in the homecages on Days 4 (prior to the first injection), 6, 8, 10, 12, and 14 of the lactation period. The number of attacks was recorded for each aggression test. Weights of the lactating animals and their litters were recorded each day. RESULTS Our informal daily observations of maternal behavior indicated that the lactating animals within each group exhibited normal maternal care including nest building, licking, retrieving, and crouching over the young. Figure 1 shows the number of attacks directed toward the stimulus intruders as a function of the dose of TP and the postpartum test day. It is evident from this portrayal that the animals of each group exhibited a decfine in the intensity of aggression with advancing lactation; the decline in fighting is not uncommon and previously has been demonstrated for mice, rats, and hamsters [6, 19, 28]. However, the figure also shows that the d e c l i n e in the n u m b e r o f attacks w a s more precipitous for the TP groups, with the high doses of the steroid more effective in s u p p r e s s i n g the behavior than l o w e r doses. Analysis of Variance Tests (ANOVA) [27] on the number of attacks yielded a significant Treatment effect, F(3,28)=6.8, p<0.005, and a significant Postpartum Test Day effect, F(5,140)=660.3, p<0.005. Further analysis using

the Honestly Significant Difference Test (HSD) [21 (c~=0.05) showed that the number of attacks exhibited by the TP groups was significantly less than that displayed by the oil control. Moreover, the 250 and 500 f g TP groups, while not differing from each other, exhibited significantly fewer attacks than animals of the 125 #g TP group. Additional individual comparisons using the HSD Test (a=0.05) revealed that the groups did not differ from each other on the first test day prior to steroid or oil treatment (Postpartum Day 4). However, with the exception of the 125/xg TP group, the TP groups exhibited significantly fewer attacks than the oil controls on each test day beginning on Postpartum Day 6 (i.e., the first test following TP treatment). The 125 f g TP group exhibited significantly fewer attacks than the oil control on Postpartum Day 6, 12, and 14. The latencies to attack in all groups were immediate (averaging less than 5 sec) and were directed primarily to the flank area of the opponent. Instead of exhibiting intense aggressive behavior, the animals administered TP appeared to be spending more time in social investigation (i.e., grooming and sniffing) than did the oil control animals. The mean of the average pup weight gain as a function of treatment group and postpartum day is depicted in Fig. 2. The unit of analysis was average pup weight gain rather than total litter weight gain because several pups died during the course of the study. The occasional loss of pups was not systematically related to any of the groups; they were replaced by like-age foster young as soon as their absence was noted. (Foster young were not included in the statistical analysis). Figure 2 shows that lactation performance, as measured by average pup weight gain, declined for all groups as the pups advanced in age. It is evident from this portrayal, however, that the TP-treated groups exhibited smaller average pup weight gains each day beginning on about Postpartum Day 8. ANOVA tests performed on the average pup weight gain showed a significant Treatment, F(3,28)= 14.6, p <0.01, Postpartum Day, F(10,280) = 14.5, p <0.01, and Treatment x Postpartum Day effect, F(30,280)=4.0, p<0.001. Further analysis of the treatment effect using the HSD Test (a=0.05) showed that the average pup weight gain for the TP-treated groups was significantly less than that for the oil controls. The 250 and 500/zg TP groups did not differ from each other but did exhibit smaller average pup weight gain than the 125/~g TP group. Individual comparisons using the HSD test (a=0.05) showed that the groups did not differ from each other on Postpartum Days 4-7. Beginning on Postpartum Day 8, however, the TP-treated animals exhibited significantly smaller average pup weight gain on each of the remaining postpartum days as compared to the oil controls. The average pup weight gain of the 500/xg TP group was significantly smaller than the 125 ~g TP group on Postpartum Days 11 and 13; the same measure for the 250/zg TP group was significantly smaller than the 125/zg TP group on Postpartum Days 10 and 13. The mean body weight of the dams as a function of treatment and postpartum day is known in Fig. 3. It is evident that the dams of all groups increased in their body weight over the course of testing. Figure 3 also shows that at the end of the treatment period the groups administered TP had lower body weights than did the oil control. An ANOVA test showed a statistically significant Postpartum Day Effect, F(I 1,308)= 16.4, p<0.001, and Treatment xPostpartum Day Effect, F(33,308)=6.5, p<0.001. Individual comparisons using the HSD Test (a=0.05) showed that the mean body weights of the TP groups were significantly lower than the oil

TESTOSTERONE AND MATERNAL AGGRESSION

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FIG. 2. The average pup weight gain per day for young nursed by lactating female mice treated daily from Postpartum Days 4-13 with oil, 125,250, or 500/zg TP.

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FIG. 3. The average body weight per day for lactating female mice treated daily from Postpartum Days 4-13 with oil, 125, 250, or 500/zg TP.

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controls on Postpartum Days 12-14. In addition, the 125/zg TP group exhibited significantly smaller body weights than those of the oil control on Postpartum Day 9 while the body weights of the 500 tzg TP group were significantly smaller than those of the oil control on Postpartum Days 10--14. Finally, the 125 and the 500/xg TP groups significantly differed with respect to body weight on Postpartum Days 11 and 13. A comparison of the aggression (number of attacks), lactation (average pup weight), and dam weight (Figs. 1, 2, and 3) reveals that deficits occurred in all three measures following TP treatment. However, it is significant to note that inhibition of maternal aggressive behavior occurred long before adverse effects on lactation and dam weights were observed. DISCUSSION The present findings indicate that TP suppresses maternal aggressive behavior in lactating female mice. Daily doses of 125, 250, or 500/~g TP were found to significantly decrease the number of attacks displayed by lactating females toward male intruders. The suppression of fighting at the 250 and 500 /xg TP doses was observed after as little as 2 injections while the 125/~g TP dose was somewhat less effective in that 4 injections were required before reduced fighting behavior was observed. These findings are similar to an earlier report showing that exogenous estrogen (E) also inhibits the behavior in mice [20]. The present findings are in general agreement with other reports demonstrating the adverse effects of TP on other aspects of maternal behavior (cf., lactation, nest building, pup killing, etc.) [5, 7, 8]. On the other hand, to our knowledge, the aggression-inhibiting nature of TP observed in this study represents the first report of the steroid suppressing instead of promoting mammalian agonistic behaviors (cf., intermale, virgin female, and predatory aggression) (cf. [9,10]). Testosterone may suppress maternal aggression by directly inhibiting the neural tissue involved in the exhibition of the behavior. In the mouse, T and E are known to be taken up in large quantity by sites in the hypothalamus and septum [15], areas of the brain known to mediate aggressive behaviors in many different species (cf. [11, 12, 16]). Because a previous report shows that E also inhibits the behavior [20], and because T can be metabolically converted (aromatized) to E in the brain, the suppression of the behavior may be primarily mediated by E. Although the above interpretation of the present findings is the most viable one at the present time, there are at least three other alternative explanations which should also be considered. First, T may have exerted

its effects by altering the mother-young interaction. Suckling stimulation is known to be a prerequisite for the initiation of the behavior in mice [22,23] and the quality or quantity of this stimulation may have been altered in our TP-treated animals. This explanation would seem to be unlikely since the maternal behavior (especially nursing) of our dams appeared to be normal. Second, if not directly affecting the receipt of suckling stimulation, TP may have depressed maternal aggression by altering lactation, the physiological consequence of suckling. Lactation, as assessed by daily pup weight gain, was depressed in our TP-treated animals and recent findings indicate that pharmacological blockade of the primary lactogenic hormone, prolactin (PRL), inhibits maternal aggression in hamsters [29]. Thus, a reduction in PRL or alterations in other lactogenic hormones (i.e., adrenal corticosterone) may have been responsible for the decline in the aggressive behavior of TP-treated dams. The above alternative also would seem to be unlikely, however, because deficits in lactation were observed long after the suppression of fighting behavior, and because our previous work has repeatedly shown that other endocrine glands, and lactation are not necessary for the initiation and maintenance of the behavior (cf. [23]). Finally, the reduced body weight of the TP-treated animals also may explain the inhibitory effect of the steroid on maternal aggression. Body weight is known to be a reliable predictor of dominance-subordinate relationships with high ranking individuals usually weighing more than low ranking animals (cf. [13,25]). Because reduced body weight following TP administration was detected long after the suppression of fighting behavior was first observed, this alternative explanation also would not seem to be a viable one. The question remains as to whether or not the observed inhibitory effect of TP on maternal aggressive behavior is a pharmacological one, or whether endogenous circulating levels of T might normally inhibit the behavior. RocklandSwiss albino mice do not exhibit aggressive behavior during pregnancy or the first 24 hr following parturition [21,22]. Because maternal plasma T levels are high in the mouse during the above reproductive stages [1], it is interesting to speculate that circulating levels of T during the gestational and early postpartal periods may be responsible for the absence of aggression. ACKNOWLEDGEMENTS The author gratefully acknowledges the assistance of Cynthia Betteridge, Debra Katz and Owen Samueis in the conduct of this research. This work was supported by U.S.P.H.S. Grant MH-32467 from N.I.M.H. and by a SUNY Research Foundation Grant.

REFERENCES

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439 19. Svare, B. and R. Gandelman. Postpartum aggression in mice: Experiential and environmental factors. Hormones Behav. 4: 323-334, 1973. 20. Svare, B. and R. Gandelman. Postpartum ac,oression in mice: Inhibitory effect of estrogen. Physiol. Behav. 14: 31-36, 1975. 21. Svare, B. and R. Gaadelman. A longitudinal analysis of materhal aggression in Rockland-Swiss albino mice. Devel Psychobiol. 9: 437-446, 1976. 22. Svare, B. and R. Gandelman. Postpartum aggression in mice: The influence of suckling stimulation. Hormones Behav. 7: 407-416, 1976. 23. Svare, B. and R. Gandelman. Suckling stimulation induces aggression in virgin female mice. Nature 320: 606-608, 1976. 24. Svare, B., P. Davis and R. Gandelman. Fighting behavior in female mice following chronic androgen treatment during adulthood. Physiol. Behav. 12: 399-403, 1974. 25. Uhrich, J. The social hierarchy in albino mice. J. comp. physiol. Psychol. 25: 373-413, 1938. 26. vom Saal, F. S., B. Svare and R. Gandelman. Time of neonatal androgen exposure influences of length of testosterone treatment required to induce aggression in adult male and female mice. Behav. Biol. 17: 391-397, 1976. 27. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 28. Wise, D. A. Aggression in the female golden hamster: Effects of reproductive state and social isolation. Hormones Behav. 5: 235-250, 1974. 29. Wise, D. A. and T. L. Pryor. Effects of ergocornine and prolactin on aggression in the postpartum golden hamster. Hormones Behav. 8" 30-39, 1977.