The progesterone challenge: steroid hormone changes following a simulated territorial intrusion in female Peromyscus californicus

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Hormones and Behavior 44 (2003) 185–198

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The progesterone challenge: steroid hormone changes following a simulated territorial intrusion in female Peromyscus californicus Ellen S. Davis* and Catherine A. Marler Department of Psychology, University of Wisconsin, Madison, WI 53706, USA Received 3 June 2002; revised 23 December 2002; accepted 21 February 2003

Abstract There is a growing body of evidence that the rapid but transient increase in male androgens, particularly testosterone (T), following a single social encounter such as a territorial intrusion occurs in a wide array of vertebrate taxa. Yet, this phenomenon, often called the Challenge Hypothesis, has rarely been investigated in females. Moreover, when studying male challenge effects, researchers have rarely investigated other hormones that can be important to the expression of aggression, such as progesterone (P4) and estradiol (E2). We conducted 10-min aggression trials using the resident–intruder paradigm in cycling female California mice, Peromyscus californicus, a species in which both sexes show territorial behavior. By comparing the hormone levels of test females to control females, we found a decrease in P4 and the P4/T ratio, but no change in T, E2, corticosterone, E2/P4, or E2/T. Interestingly, these hormone changes were observed even when the resident was not aggressive toward the intruder, suggesting that the stimulus cueing the hormone changes was the mere presence of the intruder and not the amount of aggression displayed by the resident. Generally, T has a positive relationship with aggression, whereas P4 inhibits male and nonmaternal female aggression. Thus, decreasing the P4/T ratio following an encounter may serve to increase future aggression in females. These results suggest that females may use different hormonal mechanisms than do males to mediate aggression in a challenge situation. © 2003 Elsevier Inc. All rights reserved.

There is growing evidence that the transient increase in male testosterone (T) levels following an acute challenge is a widespread phenomenon. For example, the classic studies with passerine birds found an increase in T in response to territorial intrusions by conspecific males (Wingfield, 1985, 1988). Parallel studies in humans reported changes in T in both athletes (Booth et al., 1989) and fans (Bernhardt et al., 1998) following sporting events and that the direction of hormone change may involve mood (Mazur and Lamb, 1980). Often called the Challenge Hypothesis, this intriguing interaction between hormones and behavior has spawned a good deal of subsequent research on territoriality or dominance hierarchies in other taxa (e.g., fish: Pankhurst and Barnett, 1993; lizards: Thompson and Moore, 1992; mammals: Creel et al., 1993), although passerine birds re-

* Corresponding author. Biology Department, Upham Hall, University of Wisconsin—Whitewater, Whitewater, WI 53190. E-mail address: [email protected] (E.S. Davis). 0018-506X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0018-506X(03)00128-4

main the best studied group. It is generally maintained that these elevated levels of T do not initiate the appropriate aggressive responses, but instead serve to sustain the response (Wingfield et al., 1987). The growing number of studies has led to several important modifications of the Challenge Hypothesis, including considerations of the mating system (Wingfield et al., 1990; Wingfield, 1994a) and specific life history context (e.g., Wingfield, 1994b; Wingfield et al., 1997). However, one rarely addressed consideration is the gender of the territory holder, despite the fact that females compete among each other for resources in a wide array of taxa (reviewed by Floody, 1983). If the challenge effect is an important factor in the successful maintenance of a territory or dominance hierarchy, then females with life history characteristics such as territory defense also should exhibit a similar pattern of hormonal response to a challenge. In this study we investigated the steroid hormone response of females exposed to simulated territory intrusions in order to investigate the possibility that

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the mechanisms controlling plasticity in aggression in females are similar to those described for males. Investigations into the challenge effect have typically been limited to androgens (but see Elekonich and Wingfield, 2000), but we studied additional steroid hormones as well as their ratios for several reasons. First, the relationship between T and aggression generally is less clear for females than for males (reviewed by Floody, 1983), with positive effects of T on aggression often seen only with chronic T treatment. Second, there is some evidence that E2 can influence aggression in females (Lisk and Nachtigall, 1988; Barfield, 1984; but see Vandenbergh, 1971; Payne and Swanson, 1972; Gray et al., 1978; Barfield, 1984), and that the influence of T on aggression in males may occur via central aromatization to estradiol (E2) (e.g., Cologer-Clifford et al., 1999; Toda et al., 2001). Also, progesterone (P4) has been shown to influence aggression in both males (Erpino and Chapelle, 1971; Fraile et al., 1988) and females (Erpino and Chapelle, 1971; Payne and Swanson, 1972; Gleason et al., 1979; Barfield, 1984; Fraile et al., 1988; Meisel and Sterner, 1990; Kapusta, 1998). Therefore, it is possible that any of these hormones may be involved in a Challenge-like effect in females. Finally, there is evidence the ratios of these different hormones can affect morphology (Xia and Younglai, 2000), physiology (Christeff et al., 1999; Medras and Jankowska, 1999; Ng et al., 2000), or behavior (Weber et al., 2000), including aggression (Hilakivi-Clarke et al., 1997). We therefore investigated whether the change in hormone ratios correlate better with observed aggression levels than do individual hormones alone. In designing our study, we took into account several life history characteristics that were identified as important in previous studies of the challenge effect, as these factors may also potentially bear on a female hormonal response to a challenge. For example, the challenge effect appears to be most pronounced in territorial species with monogamous or seasonally monogamous mating systems (Wingfield, 1994a). Additionally, a challenge effect that is exhibited during the breeding season can disappear during nonreproductive times of the year, even if males retain their territories (Wingfield, 1994b; Wingfield et al., 1997). Finally, increased T in response to a challenge typically is not found when male T and aggression levels are already at a maximum (Wingfield et al., 1990). Therefore, we designed our experiment so that females (1) would display territorial behavior; (2) were cycling (i.e., not immature or pregnant); but (3) were not maximally aggressive (i.e., not displaying maternal aggression). One excellent candidate for investigating a challenge effect in females is the California mouse, Peromyscus californicus. This species is both highly territorial and strictly monogamous in the field (Ribble and Salvioni, 1990; Ribble, 1991), with both males and females exhibiting high levels of aggression (Eisenberg, 1963; Bester-Meredith and Marler, unpublished data) and parental care (Dudley, 1974;

Gubernick and Alberts, 1987; Ribble and Salvioni, 1990; Bester-Meredith et al., 1999; Trainor and Marler, 2001). Unlike many mammalian species, dispersal (which presumably would be correlated with the number of conspecifics encountered) is female-biased in this species, and females apparently compete for mates (Ribble, 1992). Moreover, recent evidence indicates that P. californicus males exhibit an increase in T following an aggressive encounter (Oyegbile and Marler, unpublished data). In contrast, males of a closely related but less territorial species, P. leucopus, did not show a challenge effect (Oyegbile and Marler, unpublished data). Given that P. californicus females exhibit similar (monogamy and territoriality) or more extreme (dispersal distance) life history characteristics as male P. californicus, this species is a strong candidate for demonstrating a challenge effect in females. To simulate territorial aggression, we staged resident– intruder encounters. We used cycling females, as they are by definition in breeding condition. By using females at different stages of the estrous cycle, we also were able to investigate the hormonal response to a challenge at different levels of aggression, which is typically greater during nonestrous periods than during estrus (reviewed by Floody, 1983). Yet, none of this aggression likely would be maximal, as females of many species exhibit the highest levels of aggression when defending their offspring (reviewed by Maestripieri, 1992). Thus, we staged encounters during various stages of the estrous cycle in order to investigate whether P. californicus females exhibit a hormonal response to a challenge.

Methods Subjects We used a total of 103 nulliparous, female mice reared in a laboratory colony at the University of Wisconsin (Madison, WI). After weaning, subjects were housed in same-sex groups in standard cages (48.3 ⫻ 26.7 ⫻ 15.6 cm) and were given Purina 5001 mouse chow and water ad libitum. Colony rooms were kept under a 14L:10D light cycle with lights on at 0300 CST. Observation rooms were kept under a similar light cycle, but with lights on at 2300 CST. Mice were transferred to the observation room at least 5 days prior to testing to allow them to adjust to this 4-h clock shift. All mice in each cage were weighted before removing any from their home cage for testing and were ranked according to their weights. We used this ranking as an estimate of dominance status and interactions prior to use in this study, as relative size differences may influence aggressive social interactions in female mice (White et al., 1969). Behavioral tests were conducted under dim red light during the early part of the dark phase (between 1330 and 1500 CST). Animals were maintained in accordance with the recommendations of the National Institutes of Health Guide for

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the Care and Use of Laboratory Animals and all protocols were approved by the UW-Madison Research Animal Resources Center. Aggression tests Seventy-eight females were randomly assigned to one of two groups (encounter or control) for each part of the estrous cycle, with the caveat that neither siblings nor cage mates were used in the same group. Two days before testing, we placed the female into a large, Plexiglas observation cage, which was divided into two chambers (30 ⫻ 29 ⫻ 30 cm and 22 ⫻ 29 ⫻ 30 cm). The cage was lined with commercial aspen shavings. A running wheel and the water bottle were located in the larger chamber and food was placed into the smaller chamber. The two chambers were connected by two small openings at the bottom of the partition. Stage of estrous cycle was determined by vaginal lavage. Because the cell types are variable at some stages of estrus in this species (Gubernick, 1988), females were lavaged for several days prior to testing. However, it was necessary to minimize the number of times females were lavaged because repeated handling of this kind can reduce aggression in females of this species (Davis and Marler, unpublished data). Typically, a female was lavaged on Day 1 immediately prior to placement in an observation cage, once again on Day 2, and finally on Day 3 after the trial was conducted. Females were grouped according to their stage of estrus on Day 3 (Allen, 1922; Long and Evans; 1922; Paull and Fairbrother, 1985; Gubernick, 1988): proestrus (Pro), estrus, metestrus (Met), diestrus day 1 (D1), diestrus day 2 (D2), or diestrus day 3 or greater (D3⫹). (Length of cycle varies in this species from 5 to 20 days long, with most variation due to the number of diestrous days (Gubernick, 1988)). The ambiguity of this last category was necessary because females were often already in diestrus on the first day they were lavaged, so it was unknown which day of diestrus they were in at the time of the trial. Another 25 female P. californicus were used as intruder opponents and were neither siblings nor cage mates of the resident female. To maximize the likelihood of eliciting aggression from the resident, we used intruders that were smaller in mass than the residents (mean ⫾ SE resident advantage: 6.67 ⫾ 0.70 g). All opponents were in diestrus at the time of the encounter. Twenty-five females were used as intruders only in a trial only once, 11 were used two times, and 2 were used three times. Regression analysis showed no difference in attack latencies as a factor of the number of encounters experienced by the intruder (r2 ⫽ 0.0006, F(1,36) ⫽ 0.021, p ⫽ 0.88). Tests were conducted by placing the intruder into the unoccupied chamber of the resident’s observation cage for 10 min. All trials were videotaped and later reviewed to assess attack latency, number of attacks, time spent chasing, and total time spent in aggressive activities. For the purposes of this study, we defined an attack as either a bite or

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the onset of wrestling behavior. We also recorded the total number of attacks and the amount of time the resident spent chasing the intruder or sniffing the intruder. Additionally, we recorded the amount of time that the mice spent in separate chambers and the amount of time the resident spent grooming herself or running on the wheel during the encounter. Finally, the amount of time intruder females spent in a freezing posture, a sign of submission (Eisenberg, 1961), was also recorded for the two stages of estrus in which the highest and lowest levels of resident aggression was observed. Freezing behavior was measured to ascertain the aggression level of the trial from the point of view of the intruder and to determine whether it was likely that less overt forms of aggression occurred when residents appeared to be less aggressive. No intruder was ever observed to initiate aggressive behavior. We treated control resident females identically to encounter residents except that no intruder was placed into the observation cage. Instead, at the beginning of the trial, we opened the cage lid and placed a hand inside. Similarly, at the end of the trial we placed a hand inside the cage and stirred up some of the bedding to mimic the disturbance that often accompanies retrieval of the intruder. Thirty minutes after the end of the trial, residents were euthanized by decapitation, and trunk blood was collected in heparinized tubes. The samples were then centrifuged for 25 min, and the plasma portion was collected. Plasma samples were then immediately placed in a ⫺80°C freezer until the hormone analyses were conducted. Also, immediately following euthanasia, we measured the resident’s anogenital distance and lavaged the female to confirm the stage of estrus at the time of the trial. Brains were also harvested for analyses not reported here. Hormone assays Hormone assays were conducted at the Wisconsin Regional Primate Research Center Assay Laboratory. Plasma samples were extracted in anhydrous ethyl ether and then separated using celite chromatography (Abraham et al., 1972). External recoveries of tritiated P4, T, and E2 were run in duplicate to estimate procedural loss. Internal recoveries were used to estimate loss for corticosterone (Cort). Extracted samples were dried and then reconstituted in 4% ethyl acetate in isooctane (EA/ISO). Progesterone, T, E2, and Cort were eluted with 100% ISO, 20% EA/ISO, 30% EA/ISO, and 50% EA/ISO, respectively. Progesterone and T were assayed using enzyme immunoassay (EIA) techniques (Ginther et al., 2001). Microtiter plates (Nunc-Immuno Plate Maxisorb F96 certified; VWR Scientific, Chicago, IL) were coated with T antibody (R156, University of California, Davis; diluted 1:27,000) or P4 antibody (R4861, University of California, Davis; diluted 1:33,000) with coating buffer aliquoted in 100-␮l amounts per well. Fractions of P4 or T and their respective standards were assayed on these plates. Standard curves were created

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from eight standards each for P4 (5.0 –1000 pg) and T (1.0 –250 pg). Absorbance was read at 415 nm on a Spectromax 340 (Molecular Devices Corp., Sunnyvale, CA). Data reductions (log-logit) transformations were analyzed by weighted least-squares regression analysis and reported as ng/mL of plasma for P4 and pg/mL for T. Estradiol and Cort were assayed using radioimmunoassay (RIA) (Moore, 1986; Marler and Ryan, 1996). Portions of the sample extracts were incubated overnight with E2 antibody (Holly Hill Biological, Hillsboro, OR; diluted 1:6000) or Cort antibody (Esoterix Endocrinology, Calabasas Hills, CA; diluted 1:70,000) and tritiated hormone (10,000 counts). Bound and free counts were separated with dextran-coated charcoal. The mixture was then centrifuged, and the resulting supernatant was counted on a scintillation counter. Assays for T, E2, and Cort were previously validated for this species (Bester-Meredith and Marler, 2001; Trainor and Marler, 2001, 2002). When assay concentrations for serial dilutions of female California mouse plasma pool (200 – 3.125 ␮l, n ⫽ 7) were compared with P4 standards, computed regression lines did not differ significantly in slope (p ⬎ 0.05). The sensitivity of the P4 EIA at 90% binding was 5.0 pg. Accuracy measured at each standard curve point (5.0 –1000 pg, 5.0 ␮l pool plasma, n ⫽ 8) was 101.5 ⫾ 1.15%. Quality control pools were assayed for each hormone. The intraassay coefficients of variation were 2.2, 2.3, 5.6, and 6.2% for P4, T, E2, and Cort, respectively. The interassay coefficients of variation were 7.4, 15.1, 15.4, and 11.5 for P4, T, E2, and Cort, respectively. Statistical analyses Normality of the data was assessed using visual inspection of normal probability plots. Data were transformed as necessary to produce the straightest possible probability plots, yielding the best approximation to normality for each data set. The best transformations, using this criterion, were as follows. Data for P4 and T were log transformed. Data for E2, Cort, P4/T, E2/T, and E2/P were square-root transformed. Julian date and resident weight (two possible covariates used in the model building) were also square-root transformed. Finally, attack latency was log transformed, whereas chasing, self-grooming, and wheel running were square-root transformed. We analyzed hormonal data using analysis of covariance (ANCOVA) with both forward and backward stepwise model building techniques (Statistica 5.5; Statsoft, Tulsa, OK). This type of analysis allowed simultaneous consideration of the two main factors of interest, as we always retained treatment (encounter or control) and estrus stage as categorical variables in the model. At the same time, we were able to minimize the number of covariates in the model by retaining only those that improved the overall model. Possible covariates were Julian date, resident weight, resident age, and home cage rank by weight. Note

that many models retained only one covariate, most often Julian date. All covariates retained are reported in the results for each model. Planned comparisons were used to examine hormonal differences between encounter and control females within each part of the cycle. As some residents showed no aggression during the trial, we also reanalyzed the hormonal data, categorizing the residents as control, aggressive, or nonaggressive. Because of low sample sizes in some stages of the cycle (for example, there were no nonaggressive D3⫹ females), these analyses were performed after collapsing the data across all stages of estrus. Planned comparisons were used to examine hormonal differences across the estrous cycle. In this case, we compared the hormone levels for control females only to avoid any possible influences of the encounter. Behavioral analyses also were conducted using ANCOVA with both forward and backward stepwise model building techniques. These analyses compared only encounter females across the estrus cycle, and we again retained estrus stage as a categorical variable in the model, regardless of significance. Possible covariates were those listed above plus one additional variable: weight difference between the resident and intruder. Covariates retained in the models varied among hormones and are reported in the results. Planned comparisons were used to examine hormonal differences between encounter and control females within each part of the cycle. We also attempted to investigate whether baseline hormone levels prior to the encounter were associated with aggressive behavior. However, as our results below clearly indicated that P4 decreased following encounters (and was likewise suggestive for Cort), we considered only E2, T, and E2/T as likely to be representative of baseline hormones at the time of the encounter. To approach this question, we repeated the ANCOVA analyses outlined above and in Table 1, but excluded treatment as a factor. Thus, each hormone or hormone ratio was analyzed using stage of estrus as the only main effect, and the covariates listed in Table 1 were retained for each hormone. We then regressed several measures of aggression against the residual hormones attained from these ANCOVA analyses (i.e., against the amount of variance in the hormone level not attributable to stage of estrus or the covariates) to investigate possible correlations between hormones and aggressive behavior.

Results Hormone changes Progesterone The model that best explained the variation in P4 levels (adjusted r2 ⫽ 0.32; F(12,65) ⫽ 4.1, p ⫽ 0.0001) incorporated Julian date (F1,65 ⫽ 9.15, p ⫽ 0.004) as a covariate. Overall, P4 decreased following an encounter, but did not vary across the estrous cycle (Fig. 1; Table 1). Planned

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Table 1 ANCOVA results for hormone analyses Hormone

P4 T E2 Cort P4/T E2/P4 E2/T

Estrous stage

Stage ⫻ treatment

Treatment

Covariates

F

p

d.f.

F

p

d.f.

F

p

d.f.

2.04 4.62 0.96 1.41 2.13 0.84 0.31

0.085 0.001 0.45 0.23 0.072 0.52 0.90

5,65 5,65 5,64 5,65 5,65 5,65 5,64

6.06 0.30 0.18 3.63 4.76 3.42 0.15

0.016 0.58 0.67 0.061 0.033 0.069 0.7

1,65 1,65 1,64 1,65 1,65 1,65 1,64

1.97 2.22 0.55 0.35 2.96 0.28 1.17

0.094 0.062 0.73 0.88 0.018 0.92 0.34

5,65 5,65 5,64 5,65 5,65 5,65 5,64

Julian Julian Julian Julian Julian Rank Julian

date date date, rank date date date, weight

comparisons within each stage of estrus revealed a significant decrease following an encounter in only D3⫹ (t20 ⫽ 2.42, p ⫽ 0.018) and Pro (t14 ⫽ 3.23, p ⫽ 0.002) (Fig. 1). Planned comparisons between estrous stages for control females revealed significant differences between Met and D3⫹ (t13 ⫽ 2.16, p ⫽ 0.034), Pro (t12 ⫽ 1.99, p ⫽ 0.05), and estrus (t9 ⫽ 2.77, p ⫽ 0.008).

1). Planned comparisons (Fig 2.) within each stage of estrus revealed a significant increase in T in only D1 females (t7 ⫽ 2.23, p ⫽ 0.029). Planned comparisons between estrous stages for control females revealed significant differences between estrus and D2 (t12 ⫽ 2.25, p ⫽ 0.028), D3⫹(t13 ⫽ 2.51, p ⫽ 0.014), Pro (t12 ⫽ 2.12, p ⫽ 0.038), and Met (t8 ⫽ 2.90, p ⫽ 0.005).

Testosterone As with P4, the model that best explained the variation in T levels (adjusted r2 ⫽ 0.23; F12,65 ⫽ 2.97, p ⫽ 0.002) incorporated Julian date (F1,65 ⫽ 2.71, p ⫽ 0.10) as a covariate. There was no effect of treatment on T levels, but T changed significantly over the estrous cycle (Fig. 2; Table

Estradiol The model that best explained the variation in E2 levels (adjusted r2 ⫽ 0.06; F13,64 ⫽ 1.37, p ⫽ 0.20) incorporated Julian date (F1,64 ⫽ 3.36, p ⫽ 0.071) and resident home cage rank by weight (F1,64 ⫽ 3.97, p ⫽ 0.050) as covariates. Neither treatment nor stage of estrus was significant (Fig. 3;

Fig. 1. Log-transformed P4 levels (mean ⫾ SE) across the estrous cycle. Female P4 levels (originally measured in ng/ml) were lower in encounter females than control females overall (*p ⱕ 0.05), and this treatment effect was significant for Di3⫹ and Pro females. Sample sizes are shown above the bars.

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Fig. 2. Log-transformed T levels (mean ⫾ SE) across the estrous cycle. There were no overall differences in T (originally measured in pg/ml) between encounter and control females, although there was a significant difference for D1 females (*p ⱕ 0.05). Sample sizes are as described in the legend to Fig. 1.

Table 1). Because of the heterogeneity of variance in these data, nonparametric, Kruskal–Wallis tests were also performed to confirm the ANCOVA results (estrus effects: H5 ⫽ 4.83, n ⫽ 78, p ⫽ 0.44; treatment effects: H1 ⫽ 0.055, n ⫽ 78, p ⫽ 0.81). Planned comparisons between estrous stages for control females revealed significant differences between only D1 and D2 (t12 ⫽ 2.31, p ⫽ 0.024).

stage of estrus revealed a significant decrease in D1 (t7 ⫽ 2.44, p ⫽ 0.017), D2 (t14 ⫽ 2.40, p ⫽ 0.019), and D3⫹ (t20 ⫽ 2.90, p ⫽ 0.005). Unlike P4, planned comparisons revealed no treatment effect in Pro (t14 ⫽ 1.45, p ⫽ 0.15) (Fig. 5). Planned comparisons between estrous stages for control females revealed significant differences between only D3⫹ and estrus (t13 ⫽ 2.40, p ⫽ 0.019).

Corticosterone The model that best explained the variation in Cort levels (adjusted r2 ⫽ 0.03; F12,65 ⫽ 1.20, p ⫽ 0.30) incorporated Julian date (F1,65 ⫽ 1.56, p ⫽ 0.22) as a covariate. Neither treatment nor stage of estrus was significant (Fig. 4; Table 1). Planned comparisons revealed no significant differences within the estrous stages. Planned comparisons between estrous stages for control females revealed significant differences between only D3⫹ and Pro (t16 ⫽ 2.08, p ⫽ 0.041).

E2/P4 ratio The model that best explained the variation in E2/P4 levels (adjusted r2 ⫽ 0.02; F12,65 ⫽ 1.15, p ⫽ 0.33) incorporated only resident home cage rank by weight (F1,64 ⫽ 3.84, p ⫽ 0.054) as a covariate. Neither treatment nor stage of estrus was significant (Table 1). Planned comparisons within each stage of estrus revealed no significant treatment effects. Because of the heterogeneity of variance in these data, nonparametric, Kruskal–Wallis tests were also performed to confirm the ANCOVA results (estrus effects: H5 ⫽ 1.43, n ⫽ 78, p ⫽ 0.92; treatment effects: H1 ⫽ 2.56, n ⫽ 78, p ⫽ 0.11). Planned comparisons between estrous stages for control females revealed no significant differences.

P4/T ratio The model that best explained the variation in P4/T levels (adjusted r2 ⫽ 0.24; F12,65 ⫽ 3.03, p ⫽ 0.002) incorporated Julian date (F1,65 ⫽ 3.25, p ⫽ 0.076) as a covariate. Overall, P4/T decreased significantly following an encounter, but P4/T did not vary across the estrus cycle (Fig. 5; Table 1). However, there was a significant interaction between estrous stage and treatment. Planned comparisons within each

E2/T ratio The model that best explained the variation in E2/T levels (adjusted r2 ⫽ ⫺0.03; F15,62 ⫽ 0.81, p ⫽ 0.65) incorporated

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Fig. 3. Square root-transformed E2 levels (mean ⫾ SE) across the estrous cycle. There were no differences between encounter and control females overall or within any group. Sample sizes are as described in the legend to Fig. 1.

Julian date (F1,64 ⫽ 2.88, p ⫽ 0.094) and resident weight (F1,64 ⫽ 1.06, p ⫽ 0.31) as covariates. Neither treatment nor stage of estrus was significant (Table 1). Planned comparisons within each stage of estrus revealed a significant treatment effect in D1 (t7 ⫽ 2.77, p ⫽ 0.007). Planned comparisons between estrous stages for control females revealed no significant differences. Agonistic behavior Attack latency Overall, attack latency varied across the estrous cycle, with D3⫹ females showing the shortest latencies and Pro females showing the longest (Fig. 6; Table 2). The model that best explained the variation in attack latency (adjusted r2 ⫽ 0.43; F7,30 ⫽ 5.08, p ⫽ 0.0007) incorporated Julian date (F1,30 ⫽ 7.83, p ⫽ 0.009) and resident home cage rank by weight (F1,30 ⫽ 10.2, p ⫽ 0.003) as covariates. Pairwise planned comparison analyses between the estrous stages indicated that D3⫹ residents attacked significantly faster than either Pro (t18 ⫽ 3.74, p ⫽ 0.0008) or estrus (t18 ⫽ 3.39, p ⫽ 0.002) residents. No other pairwise comparisons were significant. Number of attacks Overall, the number of attacks exhibited by the resident did not vary across the estrous cycle (Table 2). The model

that best explained the variation in the number of attacks (adjusted r2 ⫽ 0.13; F7,30 ⫽ 1.81, p ⫽ 0.12) incorporated Julian date (F1,30 ⫽ 5.76, p ⫽ 0.023) and resident home cage rank by weight (F1,30 ⫽ 2.47, p ⫽ 0.13) as covariates. Pairwise planned comparison analyses revealed no significant differences between the estrous stages. Chasing Overall, the time that females chased intruders did not vary across the estrous cycle (Table 2). The model that best explained the variation in chasing behavior (adjusted r2 ⫽ 0.18; F6,31 ⫽ 2.31; p ⫽ 0.058) incorporated only resident home cage rank by weight (F1,31 ⫽ 5.56, p ⫽ 0.025) as a covariate. Pairwise planned comparison analyses revealed that D3⫹ residents chased intruders significantly longer than either Pro (t18 ⫽ 2.87, p ⫽ 0.007) or Met (t13 ⫽ 2.23, p ⫽ 0.032) residents. No other pairwise comparisons were significant. Total time aggressive Overall, there was no difference across the estrous cycle in the total time that residents behaved aggressively toward the intruders (Table 2). The model that best explained the variation in the total time residents spent in aggressive acts (adjusted r2 ⫽ 0.27; F7,30 ⫽ 2.94, p ⫽ 0.018) incorporated Julian date (F1,30 ⫽ 4.61, p ⫽ 0.040) and resident home

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Fig. 4. Square root-transformed Cort levels (mean ⫾ SE) across the estrous cycle. There were no differences between encounter and control females overall or within any group. Sample sizes are as described in the legend to Fig. 1.

cage rank by weight (F1,30 ⫽ 5.80, p ⫽ 0.022) as covariates. Pairwise planned comparison analyses revealed that D3⫹ residents showed significantly more aggression than either Pro (t18 ⫽ 2.73, p ⫽ 0.010) or Met (t13 ⫽ 2.40, p ⫽ 0.022) residents. No other pairwise comparisons were significant. Freezing There were 19 trials involving D3⫹ or Pro residents. Of these, 14 residents showed at least some aggression toward the intruder during the trial, and 5 showed no aggression at all. Intruders showed freezing behavior in 10 of 14 trials with aggression and no intruders showed freezing behavior in the absence of aggression. Thus, freezing behavior was more likely to occur in trials with aggression than without (Yates corrected ␹2 ⫽ 4.95, d.f. ⫽ 1, p ⫽ 0.026). Also, freeze latency of the intruder was positively correlated with attack latency of the resident (r2 ⫽ 0.50, F1,17 ⫽ 16.8, p ⫽ 0.0007), and total time freezing was correlated with the total amount of aggression during the trial (r2 ⫽ 0.49, F1,17 ⫽ 16.3, p ⫽ 0.0008). Hormone changes and aggressive behavior Aggressive behavior Eight residents showed no aggression at all toward the intruders. One of 7 D2 females, 4 of 7 Pro females, 2 of 7 Estrus females, and 1 of 2 Met females did not attack. All 3 D1

and all 12 D3⫹ females showed at least some aggression during the encounter, with all D3⫹ females actually attacking the intruder. We compared hormone levels among control (no encounter), aggressive, and nonaggressive residents, but because of low sample sizes and particularly because there were no non-aggressive D3⫹ females, we collapsed the data across all stages of estrus (Table 3). There were no significant difference among all three groups (control, aggressive, and nonaggressive residents) for T, E2. Cort, E2/P4, or E2/T, but there were overall differences for P4 and P4/T. Pairwise planned comparisons revealed several significant differences between some groups. Control females had significantly higher P4 levels than either aggressive (t69 ⫽ 2.19, p ⫽ 0.032) or nonaggressive (t47 ⫽ 2.90, p ⫽ 0.005) females. Similarly, control females had significantly higher P4/T ratios than either aggressive (t69 ⫽ 2.41, p ⫽ 0.018) or non-aggressive (t47 ⫽ 2.09, p ⫽ 0.04) females. No significant differences were found between aggressive and nonaggressive females for any hormone or hormone ratio. Thus, overall, changes in hormones following an encounter occurred regardless of the level of aggression exhibited during the encounter. Baseline hormones and aggression We regressed attack latency, time spent chasing, and total time spent displaying aggression against individual E2,

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Fig. 5. Square root-transformed P4/T levels (mean ⫾ SE) across the estrous cycle. Female P4/T levels were lower in encounter females than control females overall, and this treatment effect was significant for D1, D2, and D3⫹ females (*p ⱕ 0.05). Sample sizes are as described in the legend to Fig. 1.

T, or E2/T residuals (see Methods) for (1) all females that attacked the intruder (n ⫽ 27); (2) all females that showed some aggression during the trial even if they did not attack (n ⫽ 30); and (3) all females exposed to an intruder regardless of whether they exhibited aggression (n ⫽ 38). Of these 27 analyses, seven yielded significant results. Attack latency was positively correlated with E2 residuals for all aggressive females (r2 ⫽ 0.24, F1,28 ⫽ 9.01, p ⫽ 0.006) and for all individuals (r2 ⫽ 0.12, F1,36 ⫽ 4.92, p ⫽ 0.033). Attack latency also was positively correlated with E2/T residuals for all attacking individuals (r2 ⫽ 0.16, F1,25 ⫽ 4.88, p ⫽ 0.036), for all aggressive individuals (r2 ⫽ 0.25, F1,28 ⫽ 9.57, p ⫽ 0.004), and for all individuals (r2 ⫽ 0.13, F1,36 ⫽ 5.52, p ⫽ 0.024). Time spent chasing was positively correlated with T residuals for all attacking females (r2 ⫽ 0.16, F1,25 ⫽ 4.72, p ⫽ 0.040), and negatively correlated with E2/T residuals for all aggressive individuals (r2 ⫽ 0.14, F1,28 ⫽ 4.57, p ⫽ 0.041). All other comparisons were not significant.

best explained the variation in these data (adjusted r2 ⫽ 0.22; F8,28 ⫽ 2.26, p ⫽ 0.053) incorporated Julian date (F1,28 ⫽ 2.09, p ⫽ 0.16), resident home cage rank by weight (F1,28 ⫽ 4.04, p ⫽ 0.054), and resident age (F1,28 ⫽ 4.93, p ⫽ 0.035) as covariates. However, pairwise planned comparison analyses between the estrous stages indicated that D2 residents and their intruders spent significantly more time in opposite chambers than did D3⫹ (t17 ⫽ 2.27, p ⫽ 0.031) or Pro (t17 ⫽ 2.28, p ⫽ 0.030) residents and intruders.

Other behavioral measures

Self-grooming Overall, there was there was no significant effect of estrous cycle on the time that residents spent grooming themselves (Table 2). The model that best explained the variation in these data (adjusted r2 ⫽ 0.15; F5,32 ⫽ 2.33, p ⫽ 0.065) incorporated no covariates. Pairwise planned comparison analyses between the estrous stages indicated that D2 residents spent significantly more time grooming themselves than did D3⫹ (t13 ⫽ 2.45, p ⫽ 0.020), Pro (t13 ⫽ 2.90, p ⫽ 0.007), estrus (t13 ⫽ 2.30, p ⫽ 0.028), or Met (t8 ⫽ 2.41, p ⫽ 0.022) residents.

Location Overall, there was no difference over the estrous cycle in the amount of time that residents and intruders spent on opposite sides of the cage divider (Table 2). The model that

Wheel running Overall, there was no difference over the estrous cycle in the amount of time that residents spent wheel running during the encounter (Table 2). The model that best explained

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Fig. 6. Log-transformed attack latencies (mean ⫾ SE) of encounter females across the estrous cycle. Attack latency (originally measured in seconds) varied across the estrous cycle overall, with D3⫹ females attacking significantly faster than either Pro or Estrus females. Groups that share the same letter are not significantly different from each other. Sample sizes are as described in the legend to Fig. 1.

the variation in these data (adjusted r2 ⫽ 0.27; F7,30 ⫽ 2.95, p ⫽ 0.018) incorporated Julian date (F1,30 ⫽ 6.59, p ⫽ 0.015) and home cage rank by weight (F1,30 ⫽ 4.89, p ⫽ 0.035) as covariates. Pairwise planned comparison analyses between the estrous stages revealed no significant differences.

F6,31 ⫽ 1.63, p ⫽ 0.17) incorporated the weight difference between the resident and opponent (F1,31 ⫽ 5.59, p ⫽ 0.024) as a covariate.

Sniffing Overall, there was no difference over the estrous cycle in the amount of time that residents spent sniffing the opponents during the encounter (Table 2). The model that best explained the variation in these data (adjusted r2 ⫽ 0.09;

Progesterone challenge

Discussion

We have evidence that the female equivalent for the male Challenge effect may be a progesterone challenge. For all Table 3 Hormonal comparisons based on presence of aggressive behavior

Table 2 ANCOVA results for behavioral analyses Behavior

Attack latency Number of attacks Chasing Total time aggressive Different chambers Self-grooming Wheel running Sniffing

Hormone Transformed means ⫾ SE

Estrous stage

Aggressive (n ⫽ 30)

Covariates

F

p

d.f.

3.87 0.69 2.22 2.16 1.78 2.33 1.57 0.86

0.008 0.63 0.078 0.086 0.15 0.066 0.20 0.52

5,30 5,30 5,31 5,30 5,28 5,32 5,30 5,31

Julian date, rank Julian date, rank Rank Julian date, rank Julian date, age, rank None Julian date, rank Weight difference

P4 T E2 Cort P4/T E2/P4 E2/T

F

p

5.29 0.75 0.22 2.25 4.02 2.06 0.02

0.007 0.48 0.80 0.11 0.022 0.13 0.98

Nonaggressive Control (n ⫽ 8) (n ⫽ 40)

2.1 ⫾ 0.88a 1.6 ⫾ 0.36a 2.56 ⫾ 0.11b 2.3 ⫾ 0.07a 2.1 ⫾ 0.16a 2.30 ⫾ 0.05a 3.6 ⫾ 0.41a 3.1 ⫾ 0.75a 3.36 ⫾ 0.28a 26.8 ⫾ 2.29a 21.8 ⫾ 4.32a 30.5 ⫾ 1.68a 6.9 ⫾ 0.51a 6.3 ⫾ 0.64a 8.37 ⫾ 0.39b 0.042 ⫾ 0.006a 0.042 ⫾ 0.012a 0.030 ⫾ 0.003a 0.24 ⫾ 0.03a 0.23 ⫾ 0.07a 0.24 ⫾ 0.02a

Note. Shared letters (a or b) indicate no significant difference between groups for each hormone individually.

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females in an encounter, the most significant hormonal changes following an encounter were observed in P4 and the P4/T ratio. Moreover, these effects were found regardless of whether stage of estrus was part of the statistical model. To our knowledge, these results are only the second demonstration of a hormonal change following a single social encounter in females and the first study in males or females in which P4 was one of the hormones involved. Although P4 generally is considered to positively influence maternal aggression (reviewed by Nelson, 2000), its effect on nonmaternal female aggression is less clear. Studies using exogenous hormone treatments have shown mixed results in female rodents for P4 (positive effects: Payne and Swanson, 1972; Gleason, et al., 1979; Kapusta, 1998; negative effects: Erpino and Chapelle, 1971; Barfield, 1984; Fraile et al., 1988; Meisel et al., 1988; Meisel and Sterner, 1990). However, some specific patterns of P4 effects have emerged. For example, P4 reduces female aggression when individuals are E2 primed (e.g., Barfield 1984; Meisel et al., 1988; Meisel and Sterner, 1990), as occurs during estrus when aggression is typically low (reviewed by Floody, 1983). Also, P4 has been shown specifically to attenuate androgen-induced aggression in both males (Erpino and Chapelle, 1971) and females (Barfield, 1984), although it did not reduce male aggression induced by E2 or dihydrotestosterone (DHT) (Gravance, et al., 1996). Note also that, of the three studies cited in which P4 increased aggression, two employed a neutral arena aggression test (Payne and Swanson, 1972; Gleason et al., 1979), and there is some evidence that even within species the hormonal control of aggression varies between the resident–intruder and neutral arena paradigms both in females (Barfield, 1984) and in males (Schuurman, 1980). Therefore, perhaps most evidence to date suggests that P4 decreases nonmaternal aggression. Moreover, there is growing evidence that P4 can have rapid effects, acting through non-genomic mechanisms (reviews by Moore and Evans, 1999; Frye, 2001; Schumacher and Robert, 2002), making P4 a good candidate for quickly altering aggression levels. Based on this general conclusion, and borrowing logic long incorporated into research on the Challenge effect in males (e.g., Wingfield et al., 1987), we hypothesize that the direction of hormonal changes we found in the current study possibly may serve to increase aggression in future encounters. We know of only three other studies in which hormonal effects following a single encounter have been studied. First Broida et al. (1981) investigated prolactin changes in lactating females following an aggressive encounter but found none. More recently, Woodley et al. (2000) found that Cort increased in both male and female mountain spiny lizards (Sceloporus jarrovi) but found no change in other steroid hormones. Finally, Elekonich and Wingfield (2000) found hormonal changes in female song sparrows, Melospiza melodia, following a simulated territorial intrusion, but surprisingly, they found a decrease in both T and DHT and no behavioral effects of exogenous hormone treatment. It is

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difficult to compare female hormonal patterns between mammals and birds, but T can inhibit pregnancy-induced aggression in mice (Svare, 1980). Elekonich and Wingfield (2000) found an androgen decrease following encounters in incubating females. Although they took steps to measure territorial and not nest defense, it is possible that the decrease in T they found might be functionally analogous to the role of T in pregnancy-induced aggression in rodents. Surprisingly, in the current study both P4 and the P4/T ratio changed following an encounter, regardless of whether the residents showed aggression toward the intruder, suggesting that exposure to an intruder is sufficient to alter hormonal profiles. Most studies on the Challenge effect in males report increased measures of aggression following a simulated territorial intrusion but do not report hormonal results based on aggression levels observed during the trials, perhaps because all individuals exhibited aggression. Regardless, Wingfield (1987) suggests that the stimuli caused by the mere presence of the intruder may be sufficient to change hormone levels. Moreover, exposure to a stimulus alone can trigger physiological changes in analogous situations. For example, human sports fans showed increased T when watching the team that they supported win and decreased T when their team lost (Bernhardt et al., 1998). Similarly, T and 11-ketotestosterone increased in male cichlid fish (Oreochromis mossambicus) after merely having watched aggressive encounters between two neighboring males (Oliveira et al., 2001). In other contexts, male mice (Batty, 1978) and rats (Purvis and Haynes, 1974) show increased T in response to nontactile exposure to females. Also, in mice a single exposure to an unknown female increases the probability that a female will show aggression in future encounters even if she did not shown aggression in the initial encounter (Meisel et al., 1988; Meisel and Sterner, 1990). Together, these results suggest that aggression itself is not necessarily required to set in motion the physiological changes that may increase aggression in future encounters. It remains to be seen, then, whether nonaggressive female P. californicus become more aggressive after encounters such as we have staged here. Two issues regarding the validity of these results should be addressed. First, because the direction of hormone changes was negative, it is important to point out that these changes did not appear to be caused by stress effects, as Cort did not increase and, if anything, there was a nonsignificant trend for Cort to decrease following an encounter. Second, although the observed change in the P4/T ratio seems to have been driven largely by changes in P4, it is unlikely that this result is wholly an artifact of the significant change in P4 for two reasons. First, as mentioned above, the individual stages of estrus in which effects were seen on P4 and the P4/T ratio were not the same. In fact, D3⫹ was the only stage in which we found changes in both variables. Second, if the effect of treatment on the P4/T ratio were simply an artifact of changes in P4, then we would expect to see a similar artifactual result for the E2/P4 ratio.

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However, there was no corresponding treatment effect found for the E2/P4 ratio overall or within any stage of estrus. Hormonal changes across the estrous cycle When comparing both behavior and hormonal changes across the estrous cycle, the complexity of these interactions quickly becomes apparent. First, when looking only at the hormone changes among control females over the cycle, this species appears to lack some of the dramatic hormone changes observed in other species (e.g., Croix and Franchimont, 1975; Michael, 1976; Nequin et al., 1979), although we did see some changes. However, we sampled females only once, always relatively early in the dark cycle, and we could have easily missed short-term hormonal peaks. Thus, our results should not be considered to represent wholly the hormone changes that might occur over the estrous cycle in this species. More surprising, however, was the complex pattern of hormone changes across the estrous cycle that were observed following an encounter. Both P4 and the P4/T ratio showed a significant overall decrease following an encounter, but the effect was significant for only some stages of estrus. (Because of the very small sample size for Met females, we hesitate to draw any conclusions for that stage.) Moreover, as discussed above, females overall showed a decrease in both P4 and the P4/T ratio, regardless of whether they exhibited aggression. It is possible that these differences in hormonal changes among estrous groups are simply statistical artifacts arising from individual variation. Alternatively, it is also possible that the hormonal response to an encounter varied across the cycle because of issues specific to the cycle. For example, Pro or estrous females may have competing influences, such as preparation for sexual receptivity, that affect their hormonal responses to agonistic encounters. Conversely, diestrous females, which are not sexually receptive, would not have such competing influences. Regardless, it is likely that future studies investigating hormone– behavior interactions across the estrous cycle will be highly fruitful. Baseline hormones and aggression We could not reasonably investigate the role of baseline P4 on aggressive behavior, as this hormone clearly seems to change following encounters. However, as there is less indication that E2, T, or their ratio changed following an encounter, we could investigate their relationships with variation in aggression observed during the encounters. Overall, we found evidence that baseline T, E2, or the E2/T ratio were associated with varying measures of aggression, with the most consistent result being that the E2/T ratio was negatively associated with attack latency. These results suggest that T promotes and E2 inhibits baseline aggression in female P. californicus. In females T often positively affects

aggression (vom Saal et al., 1976; Barkley and Goldman, 1977; Gray et al., 1978; Gleason et al., 1979; Barfield, 1984; Simon et al., 1984; Bronson et al., 1996; Kapusta, 1998), although not always (Vandenbergh, 1971; Payne and Swanson, 1972). However, a superphysiological dose of T or chronic hormone treatment was often necessary for T to have any effect in females. There also is little evidence for an effect of E2 on aggression (positive: Barfield, 1984; negative: Lisk and Nachtigall, 1971; no effect: Vandenbergh, 1988; Payne and Swanson, 1972; Gray et al., 1978; Barfield, 1984). Perhaps the E2/T ratio might be more important in explaining baseline levels of nonmaternal female aggression than either hormone alone. Regardless, if E2, T, or their ratio is involved in baseline aggression in P. californicus, then perhaps different hormonal mechanisms may be responsible for baseline levels of aggression and changes in aggression in response to social stimuli.

Summary There were several novel findings in our study. First, like males of many species, females showed a hormonal response following an encounter compared to control females. Second, unlike males, most of the hormonal changes involved P4 or the P4/T ratio rather than T. We hypothesize that females may modulate P4 and the P4/T ratio in response to territorial intrusions in order to alter aggression in future encounters. We also found evidence that other hormones may be involved in baseline levels of aggression and hypothesize that baseline and elevated levels of aggression may be controlled by different hormonal mechanisms. Overall, these results represent the first demonstration of hormonal changes in females following an encounter in which the direction of hormonal change is consistent with the idea of facilitating increased future aggression.

Acknowledgments We are indebted to David Abbott, Toni Ziegler, and Daniel Wittwer of the Wisconsin Regional Primate Research Center Assay Laboratory for assistance with the hormone assays. We thank Patricia Martin and Janet BesterMeredith for technical assistance and Zachary Sauer for help with data collection and animal care. Helpful comments on this manuscript were provided by Brian Trainor, Yvon Delville, and two anonymous reviewers. This research was funded by NSF Grants IBN-9703309 and IBN0110625 to C.A.M., by NIMH NRSA Grant MH64280-02 to E.S.D, and in part by NIH Grant RR00167 to the National Primate Research Center at the University of Wisconsin— Madison. This paper is WRPRC publication 42-019.

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