Postpartum aggression in mice: Inhibitory effect of estrogen

Physiology and Behavior, Vol. 14, pp. 31-35. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Postpartum Aggression in Mice: Inhibitory Effect of Estrogeff BRUCE SVARE AND RONALD GANDELMAN 2

Department o f Psychology, Rutgers University New Brunswick, New Jersey 08903 (Received 17 July 1974)

SVARE, B. AND R. GANDELMAN. Postpartum aggression in mice: inhibitory effect of estrogen. PHYSIOL. BEHAV. 14(1) 31-35, 1975. -- The daily administration of estradiol benzoate (EB) to lactating mice that previously exhibited aggression toward a male intruder reduced the amount of time subsequently spent fighting as well as the total number of animals that fought. EB treatment also depressed body weight of the dams and their lactation performance. Daily progesterone treatment only reduced lactation performance. Mice

Lactation

Aggression Estradiol benzoate

Progesterone

MICE exhibit aggression toward strange conspecifics of either sex during the lactation phase of the reproductive cycle [7, 10, 18, 20]. Maternal or postpartum aggression consists of intense biting attacks directed at the neck and flanks of the intruder with a latency usually of a few seconds. In contrast to the voluminous body of information pertaining to the variables that modify other forms of agonistic behavior such as intermale aggression, relatively little is k n o w n concerning the factors that can modulate postpartum aggression. Since ovarian secretory patterns undergo a profound change during pregnancy and lactation, we chose to study the influence of estrogen upon the fighting behavior of lactating mice. Estrogen previously has been shown to inhibit several forms of rodent aggressive behavior including intermale aggression in mice [1,19], maternal aggression of lactating rats toward frogs [5], and mouse killing by rats [ 12]. In the following study estrogen is administered daily to lactating mice that previously exhibited aggressive behavior.

Procedure Screening. Screening of animals for postpartum aggression was necessary because only 6 0 - 6 5 % of R-S mice exhibit such behavior. Therefore, on the 3rd postpartum day (litters had been reduced to 6 pups on the day of parturition) an adult male R-S mouse that had been group-housed (6 per cage) was introduced into the homecage of each dam 15 min following the removal of its young. The young were removed to prevent their being trampled upon during the fighting test. This period of pup removal, which was also used in the subsequent tests, has been shown not to influence postpartum aggression [7]. An animal was scored as exhibiting fighting if it bit and chased the intruder during a 3 min test. The intruder animal was removed as soon as a fight was observed. It should be noted that intruder animals rarely if ever attack lactating animals or fight back in response to an attack. Treatments and testing. Thirty-two lactating animals that exhibited postpartum aggression during the screening tests were divided into 4 groups of 8 animals, each containing approximately equal numbers of primiparous and multiparous mice. The groups of mice were treated as follows: (1) 0.1 ug estradiol benzoate (EB), (2) 0.5 ug EB, (3) I mg progesterone (P), (4) oil. Since R-S female mice kill young after daily treatment with 50 ag doses of EB [8], we employed doses of EB well below those found to induce pup-killing. If EB was shown to influence postpartum aggression it would be necessary to establish whether the effect was specific to estrogen or whether the effect could have been produced by any steroid. For that

METHOD

Animals Pregnant primiparous and multiparous Rockland-Swiss albino mice (R-S), maintained as an outbred strain in a closed colony, were housed individually in 11 x 7 x 5 in. translucent cages, the floors of which were covered with wood shavings. The mice had free access to food (Purina Laboratory Chow) and water and were maintained on a 12[12 hr light/dark cycle with lights on between 6 a.m. and 6 p.m. Testing began at 8 a.m.

t This research was supported in part by funds from the Biomedical Science Support Grant from U.S.P.H.S. and by Grant MH-06863 from N.I.C.H.D., N.I.H. 2Requests for reprints should be addressed to R. Gandelman, Department of Psychology, Rutgers University, New Brunswick, New Jersey 08903, U.S.A. 31

32

SVARE AND GANDELMAN

reason a group of animals was treated with a relatively high dose of progesterone. The h o r m o n e s were dissolved in 0.05 cc sesame oil and administered subcutaneously once a day for 12 days beginning on the 4th p o s t p a r t u m day. The oil control group was given an equal v o l u m e of the vehicle. Three-minute aggression tests were c o n d u c t e d at 3 day intervals in the homecages on Day 4 (prior to the first injection), 7, 10, 13, and 16 of the lactation period. A group-housed male intruder was used for each test. The tests are referred to in the t e x t and figures as Test 1, 2, 3, 4, and 5. The latency to the initial attack and the total time spent fighting (i.e., biting and chasing the intruder) were recorded for each of the 5 tests. In addition, weights of the lactating animals and their litters were recorded immediately after each test. RESULTS Parity did not influence any of the measures taken. Thus, the p r i m i p a r o u s / m u l t i p a r o u s distinction has not been included in the data analysis. Daily informal observations indicated that all o f the lactating animals exhibited maternal behavior. The dams frequently were observed building nests, licking, retrieving, and crouching over their young. Table 1 gives the p r o p o r t i o n of animals of each group that fought the intruder on each test. As can be seen, the groups of animals administered oil, P, or 0.1 ~g EB did not change with respect to the p r o p o r t i o n of animals fighting across test days and did not differ b e t w e e n each o t h e r on any test day. However, the group given 0.5 ug EB declined

I-.-

TABLE 1 THE PROPORTION OF LACTATING MICE TREATED DAILY WITH OIL, PROGESTERONE (P), 0.l #g ESTRADIOL BENZOATE (EB), OR 0.5 ~g EB THAT FOUGHT AS A FUNCTION OF THE DAY OF TESTING. TESTING TOOK PLACE AT 3-DAY INTERVALS BEGINNING ON THE 4TH DAY OF LACTATION. INJECTIONS WERE BEGUN IMMEDIATELY F O L L O W I N G THE F I R S T TEST. EACH GROUP WAS COMPOSED OF 8 ANIMALS.

Treatment

1

2

Day of Testing 3

4

5

Oil P (1 mg) EB (0.1 ~g) EB (0.5 ~g)

8/8 8/8 8/8 8/8

8/8 8/8 8/8 8/8

8/8 8/8 6/8 5/8

7/8 7/8 6/8 1/8

7/8 8/8 7/8 2/8

significantly in the p r o p o r t i o n of animals fighting across test days (Cochran's test, x2 = 21.4, dr= 4, p < 0 . 0 0 1 ) . The group given 0.5 ug EB differed in terms o f the n u m b e r of animals fighting from all groups on Test 4 and 5 (Fisher's test, p < 0 . 0 5 for each comparison). Animals that stopped fighting usually engaged in mutual ano-genital sniffing with the intruder. Figure 1 depicts the time spent fighting for those lactating animals that exhibited aggression. It must be noted that aggression normally wanes during the latter

30

II Oil

25

I

!1

BB EB (0.1 ug)

P(lmg) 0

EB (0.5 ug)

Z

UJ

= 2o'!'

ILl ~

I---z

51, 10

III

5

1

2

,. TEST

4

5

DAY

FIG. l. The average time spent fighting out of 3 min tests for those animals that exhibited aggressive behavior. The mice were administered oil, progesterone, or estradiol benzoate.

ESTROGEN AND POSTPARTUM AGGRESSION portion of the lactation period [7, 10, 18, 20]. This accounts, in part, for the fact that all groups spent less time fighting as the lactation period advanced. T-tests for related measures comparing the first and last test days indicated a significant decline in the time spent fighting for the group administered P (T = 7.88, d.f = 8, p<0.001), 0.1 t~g EB (T = 6.42, d f = 7, p<0.05), and oil (T = 2.94, d f = 6, p<0.05). The group given 0.5 t~g EB also showed a decline but the small number of animals that fought on the last 2 test days precluded a statistical analysis. T-tests revealed that none of the groups differed with respect to the time spent fighting on the 1st test day. However, by the 2nd test day the group given 0.5 ~g EB spent significantly less time fighting than did the group given oil (T = 3.93, d f = 14, p<0.01), P (T = 3.97, d f = 14, p<0.01), and 0.1 vg EB (T = 2.75, d f = 14, p<0.05). Similar comparisons on Day 3 of testing showed that the 0.5 vg EB and the 0.1 ug EB group spent significantly less time fighting than did the oil (T = 2.23, d f = 11, p<0.05 and T = 2.40, d f = 11, p<0.05, respectively) and P (T = 2.29, d f = 11, p<0.05 and T = 2.47, d f = 12, p<0.05, respectively) groups but did not differ from each other. As anticipated, all comparisons on Day 4 and 5 of testing were not significant owing to the low level of agression exhibited by animals of all treatment groups. The mean of the average pup weights for animals of each of the 4 groups is shown in Fig. 2. Average pup weight rather than total litter weight was used as the unit of analysis because several pups died between the 4th and 5th tests. The loss of pups was not systematically related to any of the treatment conditions and no more than one pup was lost from any one litter. Lost pups were replaced by likeage foster young as soon as their absence was discovered. Foster young were not included in the lactational performance data. As shown in Fig. 2, the average pup weight increased across all or part of the testing period for all groups. However, it is evident that the steroid-treated groups had lower average pup weights than did the oil-treated group on Test Days 3 - 5 (Lactation Days 10-16). An analysis of variance performed on the average weight of the pups revealed a significant Treatment, F ( 3 , 2 8 ) = 11.35, p<0.001; Day, F(4,112) = 1023, p<0.001, and Treatment x Day effect, F(12,112) = 37.22, p < 0 . 0 0 1 . Individual comparisons using Fisher's Least Significant Difference Test [22] with level of significance set at a = 0.05 indicated that the average pup weights of each of the steroid-treated groups were significantly smaller than those of the oil-treated group on the 3rd, 4th, and 5th test. Furthermore, the average pup weights of the 0.5/~g EB group were significantly smaller than those of the P and 0.1 vg EB groups on the 3rd, 4th, and 5th tests, while the pup weights of the 0.1 ug EB mice were significantly smaller than those of the group given P on the 4th and 5th test. The mean body weight of the dams is presented in Table 2. They increased for all groups over the course of testing. However, the groups given EB had lower body weights than did the groups given P and oil toward the end of the treatment period. An analysis of variance revealed a statistically significant Day Effect, F(4,88) = 21.27, p<0.001, and a significant Treatment x Day interaction, F(12,88) = 2.72, p<0.O01. The Treatment Effect was not significant. Individual comparisons using Fishers Test with the level of significance set at a = 0.05 showed that the mice given 0.5 #g EB had significantly lower body weights than did the mice injected with oil on Tests 3, 4, and 5. The

33

8.5

8.0

•.-.cOil o--o

,,'*'~

,os~

P

75

• ........• EB (0.1 ug)

7.0

o,,--,o

EB(0.5ug)

6.5 6.0 5.5

5.0 4.5

i •~ " / / #,#po -............. • s~" .....• • f -,"'"

/ ~ o

O ~ o

4.0 3.5 ~ 3.0 "# 1

. 3

2

TEST

. 4

I 5

DAY

FIG. 2. The mean pup weight of the litters of animals administered oil, progesterone, or estradiol benzoate. Each group was composed of 8 dams, each nursing 6 pups. TABLE 2 MEAN BODY WEIGHTS (IN GRAMS) OF LACTATING MICE ADMINISTERED OIL, PROGESTERONE (P), 0.1 ~g ESTRADIOL BENZOATE (EB), OR 0.5/.tg EB. EACH GROUP WAS COMPOSED OF 8 ANIMALS.

Treatment

1

2

Day of Testing 3

4

5

Oil P (1 mg) EB (0.1 t~g) EB (0.5 ug)

43.6 44.8 43.7 44.0

44.8 45.0 45.8 44.9

46.0 45.3 45.3 44.2

47.0 47.5 46.1 44.6

47.9 48.4 46.2 44.9

animals given 0.5 t~g EB also had significantly lower body weights than the animals that received P and 0.1 ug EB on the 4th and 5th test. Finally, the 0.1 vg EB group exhibited significantly lower body weights than did the P and oil group on the last day of testing. Spearman rank order correlations among the time spent fighting, lactational performance, and dam weights for each group on each test day showed that none of the measures were significantly related to each other. DISCUSSION The administration of EB can inhibit the display of postpartum aggression in mice. Both dosages of EB were found to significantly decrease the amount of time that lactating animals spent fighting male intruders. A daffy dose of 0.5 ug EB was more effective than a 0.1 ~g dose in that the former significantly depressed aggressive behavior after only 3 injections as opposed to 6 injections for the latter.

34

SVARE AND GANDELMAN

Furthermore, animals in the 0.5 ug EB group completely stopped fighting after 9 injections while animals in the 0.1 ~g EB group fought throughout the course of testing. That maternal aggression was not inhibited by the 1 mg dose of progesterone indicates that the attenuation of fighting was specific to estrogen. This finding also indicates that maternal aggression and intermale aggression are differentially sensitive to progesterone since it has been demonstrated that progesterone (in dosages less than 1 mg) inhibits androgen-activated intermale fighting in mice [6,13]. In addition to its effectiveness in suppressing postpartum aggression of mice, estrogen, as mentioned previously, also has been reported to attenuate other forms of rodent aggressive behavior [5, 12, 19]. However, little is known concerning its site or mechanism of action. The inhibitory effect of estrogen on aggressive behavior may be related to the fact that estrogen is taken up in large quantity by sites in the hypothalamus [ 17], an area of the brain known to modulate agonistic behavior in several species [ 16]. There are at least four possible ways in which estrogen may influence postpartum aggression. First, estrogen may alter the display of postpartum aggression by activating a competing behavior such as sexual receptivity. Lordosis in the rat can be activated by chronic low doses of estrogen in the absence of progesterone stimulation [4]. This explanation is unlikely, however, since sexual behavior was never observed in any of the EB-treated animals. Second, it is possible that estrogen produced its effect on aggression by subtly altering the maternal behavior of the dams. This explanation is also unlikely since our observations indicated that the EB-treated animals displayed normal maternal activities. Finally, it is more plausible that estrogen exerted its influence either by inhibiting neural tissue involved in the display of aggression or, perhaps, by altering the secretory patterns of other endocrine glands such as the pituitary and the adrenal [ 1 1 ]. It also was shown that both EB and P inhibit lactation performance. Since 0.5 ug EB exerted a depressive effect upon postpartum aggression prior to the time that it depressed lactatation and since P depressed lactation (albeit

to a lesser extent than did EB) but did not influence fighting, it appears that the depressed lactation performance did not mediate the reduction in fighting behavior. The lack of a significant correlation between fighting behavior and lactational performance further supports this notion. The present data confirm previous findings concerning the depressive action of estrogen upon lactation [2,14]. Recent evidence indicates that estrogen inhibition of lactation occurs peripherally at the mammary gland [3]. Estrogen may compete with other hormones for receptor sites within the mammary gland [ 15] and/or estrogen may abolish the milk ejection reflex by blocking the action of oxytocin on the alveoli and ducts [3]. The finding that progesterone also depressed lactation performance was surprising. To our knowledge, this represents the first report of progesterone producing such an affect in a laboratory animal. However, the previous studies that have examined the effects of progesterone upon lactation have used rats [2,9] which suggests that species differences may account for the effect reported here. The suppressive effect of estrogen upon body weight of the lactating animals supports previous work concerning the influence of gonadal steroids on food regulation (cf. [21 ]). One could argue that the effect of EB upon maternal aggression was mediated through a change in body weight. This seems unlikely, however, since the lactating animals showed a decline in fighting behavior long before the steroid exerted its effect upon body weight and since fighting behavior and body weights of the dams were not correlated. At this time it is not clear whether the inhibitory effect of estrogen upon postpartum aggression is a pharmacological one, produced by hyperphysiological levels of the steroid or whether endogenous levels of estrogen normally inhibit aggressive behavior. We have recently observed that lactating mice do not exhibit aggressive behavior during the first 48 hr following parturition (unpublished data). It is possible that the sharply rising level of estrogen which occurs just prior to parturition [23] is responsible for the lack of aggression.

REFERENCES 1. Banerjee, U. Influence of some hormones and drugs on isolation-induced aggression in male mice. Communs Behav. Biol. 6: 163-170, 1971. 2. Ben-David, M., H. Roderig, K. Khazen and F.G. Sulman. Effect of different steroids on lactating rats. Proc. Soc. exp. Biol. Med. 120: 620-622, 1965. 3. Bruce, J. D. and V. D. Ramirez. Site of action of the inhibitory effect of estrogen upon lactation. Neuroendocrinology 6: 19-29, 1970. 4. Davidson, J. M., E. R. Smith, C. H. Rodgers and G. J. Bloch. Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3: 227-229, 1968. 5. Endroczi, F., K. Lissak and G. Telegdy. Influence of sexual and adrenocortical hormones on the maternal aggressivity. Acta physiol, hung. 14: 353-357, 1958. 6. Erpino, M. J. and T. C. ChappeUe. Interactions between androgens and progesterone in mediation of aggression in the mouse. Hormones Behav. 2: 265-272, 1971. 7. Gandelman, R. Mice: Postpartum aggression elicited by the presence of an intruder. Hormones Behav. 3: 23-38, 1972.

8. Gandelman, R. Maternal behavior in the mouse: Effect of estrogen and progesterone. Physiol. Behav. 10: 153-155, 1972. 9. Herrenkohl, L. R. Effects on lactation of progesterone injections administered after parturition in the rat. Proc. Soc. exp. Biol. Med. 138: 39-42, 1971. 10. King, J. A. Maternal behavior and behavioral development in two subspecies of Peromyscus maniculatus. J. Mammal. 39: 177-190, 1958. 11. Kitay, J.I. Effects of estrogen and androgen on the adrenal cortex of the rat. In: Functions o f the Adrenal Cortex, 1Iol. 2, edited by K. W. McKerns. New York: Appleton-CenturyCrofts, 1968. 12. Leaf, R.C., L. Lerner and Z.P. Horowitz. Role of the amygdala in the pharmacological and endocrinological manipulation of aggression. In: Aggressive Behavior, edited by S. Garattini and E. B. Sigg. New York: John Wiley and Sons, 1969. 13. Luttge, W.G. Activation and inhibition of isolation induced intermale fighting behavior in castrate male CD-1 mice treated with steroidal hormones. Hormones Behav. 3: 71-81, 1972.

ESTROGEN AND POSTPARTUM AGGRESSION 14. Meites, J. and J. T. Sgouris. Can the ovarian hormones inhibit the mammary response to prolactin? Endocrinology 53: 17-23, 1953. 15. Mizuno, H. and N. Sensui. A counteraction of cortisol on lactation depressing effect of estradiol in the mouse. Endocr. Jap. 20: 167-174, 1973. 16. Moyer, K. E. Kinds of aggression and their physiological basis. Communs Behav. Biol. 2: 65-87, 1968. 17. Pfaff, D.W. Autoradiographic localization of radioactivity in rat brain after injection of tritlated sex hormones. Science 161: 1355-1356, 1968. 18. St. John, R. D. and P. A. Coming. Maternal aggression in mice. Behav. Biol. 9: 635-639, 1973.

35 19. Suchowsky, G. K., L. PergIassi and A. Bonsignori. Steroids and aggressive behavior in isolated male and female mice. Psychopharmacologia 21: 32-38, 1971. 20. Svare, B. and R. Gandelman. Postpartum aggression in mice: Experiential and environmental factors. Hormones Behav. 4: 323-334, 1973. 21. Wade, G.N. Gonadal hormones and behavioral regulation of body weight. Physiol. Behav. 8: 523-534, 1972. 22. Wirier, B. ]. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1971. 23. Yoshinaga, K., R.A. Hawkins and J.F. Stocker. Estrogen secretion by the rat ovary in vivo during the estrus cycle and pregnancy. Endocrinology 85: 103-112, 1969.