Temporal relationships between shock treatment and intermale fighting behaviour in laboratory mice. Effects of modifying testicular and adrenal functions

The three experiments of this series were conducted to (1) examine subsequent effects of electric shock on intermale fighting in previously isolated mice, and (2) determine to what extent post-shock fighting is affected by manipulations of the pituitary-gonadal and pituitary-adrenocortical axes. In experiment 1 it was found that shock treatment significantly increases fighting $\text{1}\text{2}$ to 1 h later. Experiment 2 showed that castration alone does not affect post-shock fighting, whereas adrenalectomy and castration combined with adrenalectomy both inhibit such fighting. Experiment 3 showed that pretreatment with testosterone increases fighting in intact males, but fails to restore post-shock fighting in adrenalectomized and castrated-adrenalectomized males. These findings suggest that (1) the pituitary-adrenocortical axis plays an important role in controlling post-shock fighting in mice, and (2) the inhibition of post-shock fighting by adrenalectomy and castration combined with adrenalectomy is not a result of the loss of adrenal testosterone which accompanies these operations.     

Role of the pituitary-adrenocortical axis in the control of agonistic behaviour after single and repeated footshock in castrated male mice

Two experiments were conducted to examine the role of the pituitary-adrenocortical axis in the mediation of the effects of single and repeated electric footshock on subsequent agonistic responding in castrated male mice. It was found that (1) preventing corticosterone responses to shock occludes the facilitatory effects of single shock on both aggressive and submissive behaviour and occludes the additional increases in submissive behaviour which normally occur after repeated shock, and (2) blocking pituitary release of ACTH by dexamethasone treatment restores aggressive behaviour after repeated shock, independently of the initial levels of corticosterone and testosterone. These findings suggest that (1) increases in aggressive and submissive behaviour in response to single shock depend on shock-induced increases in corticosterone levels; (2) further increases in submissive behaviour in response to repeated shock require further increases in the corticosterone levels; and (3) inhibition of aggressive behaviour after shock repetition appears to depend on increases in ACTH levels, and occurs independently of the initial corticosterone and testosterone levels.     

Brief Exposure to Female Odors “Emboldens” Male Mice by Reducing Predator-Induced Behavioral and Hormonal Responses

In rodents, where chemical signals play a particularly important role in determining intersexual interactions, various studies have shown that male behavior and physiology is sensitive to female odor cues. Here we examined the effects of brief (1 min) and more prolonged (60 min) preexposure to the odors of a novel estrous female on the behavioral and hormonal responses of sexually experienced and inexperienced male mice, Mus musculus, to subsequent predator (cat and weasel) odor exposure and potential predator risk. Brief, but not prolonged, preexposure to the odors of an estrous female decreased the aversion and avoidance responses of male mice to cat odor in a Y-maze preference test, with the extent of responses being affected by a males prior sexual experience. Similarly, brief, but not prolonged, preexposure to female odors markedly attenuated the analgesic responses elicited in male mice by weasel odor. Brief exposure to a novel estrous female by itself had no significant immediate effects on either corticosterone or testosterone levels in the males. However, brief, but not prolonged, preexposure to the odors of an estrous female attenuated the marked increase in corticosterone and decrease in testosterone that were induced in males by exposure to weasel odor. The decreases in aversive responses to, and effects of, predator odor exposure that are induced by brief exposure to a novel estrous female may reflect a greater risk taking and boldness in males that could directly facilitate access to an immediately, and possibly transiently, available novel sexually receptive female.     

Hormones, sexual signals, and performance of green anole lizards (Anolis carolinensis)

The evolutionary processes that result in reliable links between male signals and fighting capacity have received a great deal of attention, but the proximate mechanisms underlying such connections remain understudied. We studied a large sample of male green anole lizards (Anolis carolinensis) to determine whether testosterone or corticosterone predicted dewlap size and/or bite-force capacity, as dewlap size is known to be a reliable predictor of bite-force capacity in territorial males. We also examined whether these relationships were consistent between previously described body size classes ("lightweights" and "heavyweights"). Heavyweights had 50% higher testosterone concentrations than lightweights during the breeding season, suggesting a mechanism for the disproportionately larger heads and dewlaps and higher bite-forces of heavyweights. Plasma testosterone concentrations were positively correlated with dewlap size and bite-force performance in lightweights (but not heavyweights) but only because of mutual intercorrelation of all three variables with body size. We suggest two possibilities for the relationship between testosterone levels and body size: (1) testosterone promotes growth in this species or (2) smaller sexually mature males are unable to compete with larger males such that the benefits of elevated testosterone do not outweigh the costs. Corticosterone levels did not differ between the male morphs, and lightweights, but not heavyweights, showed an inverse relationship between testosterone levels and corticosterone levels. Our results suggest that testosterone is important for traits related to dominance in adult male green anoles and may influence the ability to compete with rivals via fighting ability or through the use of signals.     

Activation and inhibition of isolation induced inter-male fighting behavior in castrate male CD-1 mice treated with steroidal hormones

Intermale fighting behavior between castrate male CD-1 mice living in isolation and castrate male CD-1 mice living in groups of 10 was activated by treating the isolated males with either testosterone or testosterone propionate. Fighting behavior was not activated by treating isolated males with androstenedione or dihydrotesterone even though these androgens were active in maintaining seminal vesicle weight at levels equal to, or greater than, those observed in gonadally intact males. Neither fighting behavior nor seminal vesicle weight was stimulated by treatment with progesterone, estradiol benzoate, or estradiol benzoate plus progesterone. Concurrent administration of progesterone inhibited fighting behavior activated by low, but not moderate to high doses of testosterone propionate. These results suggest that the hormone requirements for activation of fighting behavior in the male CD-1 mouse are more restrictive than those for maintenance of peripheral target tissues and that progesterone acts in the brain to competitively inhibit androgen-activated fighting.     

The endocrine stress-response and social status in the wild baboon

The relationship between social status and the testosterone and cortisol stress responses was studied in male olive baboons living in their natural environment in Kenya. (1) A variety of measures of social status are correlated with each other but are not correlated with aggressiveness or frequency of fighting. (2) Aggressiveness is positively correlated with high testosterone titers. (3) In contrast, copulatory success is not correlated with testosterone titer. Instead, it is associated with the change of testosterone levels with time; successful males increase testosterone titers in response to stress while subordinates show declines. (4) Finally, those same males with high rates of copulation showed the lowest initial cortisol levels but showed relatively faster and greater cortisol elevations following stress.     

Effects of testosterone and corticosterone on immunocompetence in the zebra finch

The original immunocompetence handicap hypothesis (ICHH) suggested that testosterone has a handicapping effect in males by both promoting the development of sexual signals and suppressing immune function. A modified version, the stress-linked ICHH, has recently proposed that testosterone is immunosuppressive indirectly by increasing production of corticosterone. To test both the original and stress-mediated versions of the ICHH, we implanted male zebra finches taken from lines selected for divergent maximum stress-induced levels of corticosterone (high, low and control) with either empty or testosterone-filled implants. Their humoral and cell-mediated immune responses were then assessed by challenge with diphtheria:tetanus vaccine and phytohemagglutinin respectively. We found no effect of the hormone manipulations on either PHA or tetanus antibody responses, but found a significant interaction between titers of both testosterone and corticosterone on diphtheria secondary antibody response; antibody response was greatest in individuals with high levels of both hormones. There was also a significant interactive effect between testosterone treatment group and corticosterone titer on body mass; the body mass of males in the elevated testosterone treatment group decreased with increasing corticosterone titer. These results suggest that, contrary to the assumption of the stress-mediated version of the ICHH, high plasma levels of corticosterone are not immunosuppressive, but are in fact immuno-enhancing in the presence of high levels of plasma testosterone. Equally, the central assumption of the ICHH that testosterone is obligately immunosuppressive is also not supported. The same individuals with the highest levels of both hormones and consequently the most robust antibody response also possessed the lowest body mass.     

The influence of glucocorticoids during the neonatal period on the development of play-fighting in Norway rat pups

A series of experiments was designed to investigate the influence of glucocorticoids on the development of play-fighting in rat pups. Previously we have found male-typical high levels of play-fighting to depend on the presence of androgens in neonatal life. Here we report that neonatally administered glucocorticoids act to suppress these high levels of play-fighting in males. In Experiment 1, male neonates treated on either Days 1 and 2 or Days 3 and 4 of life with 300 μg of corticosterone play-fought less frequently than did oil-treated animals. Corticosterone treatment on Days 9 and 10 of life had no effect suggesting that there is a “critical period” for the corticosterone effect. Similar corticosterone treatment of female pups did not influence the frequency of play-fighting. In Experiment 2, 300 μg dexamethasone, administered on Days 3 and 4 of life, had an effect in males, comparable to corticosterone. These results suggest that there is a sex-dependent, organizational effect of glucocorticoids on the development of play-fighting in rat pups. Additional experiments showed that corticosterone treatment of males on Days 1 and 2 of life did not affect adult male sexual behavior nor did it affect levels of circulating testosterone measured on Day 3 of life. These results suggest that glucocorticoid inhibition of testosterone secretion cannot account for the effect on play behavior. The possibility that glucocorticoids act directly on neural tissues to counteract testosterone effects is discussed.     

Sex differences in behavioral and corticosterone responses to mild stressors in ICR mice are altered by ovariectomy in peripubertal period

Among rodents, females are generally considered to be highly responsive in terms of emotionality under stressful conditions, and have higher corticosterone levels and activity. In this study, we examined sex differences in mice by evaluating anxiety behaviors and corticosterone responses to mild stressors. In our first experiment, we analyzed the behavioral and corticosterone responses to the elevated plus-maze test and open-field test in male and female mice, and compared sex differences. Principal component analysis (PCA) was used to investigate the correlation of these responses between males and females. The corticosterone level was higher in females under both basal and stressed conditions. In the behavioral response, higher locomotor activity was seen in females in the elevated plus-maze test. PCA showed little association among anxiety behavior, locomotor activity, and corticosterone secretion. In our second experiment, we examined the activational effects of sex steroids on the corticosterone response to the elevated plus-maze test by gonadectomizing male and female mice and using testosterone or estrogen capsules as hormonal replacements. Sex differences at the basal corticosterone level were not altered by the hormonal milieu in adults, however the higher corticosterone level of females in response to stress was diminished by ovariectomy, although replacement with neither testosterone nor estrogen had any effect. These results suggest that the sex difference in novelty exposure observed in the form of a greater hypothalamic-pituitary-adrenal (HPA) axis response in female ICR mice is controlled by ovary-derived factors in adults.     

The role of parasite-induced immunodepression,rank and social environment in the modulation of behaviour and hormone concentration in male laboratory mice (Mus musculus).

Peripheral immune responsiveness in male laboratory mice was reduced by infection with the trichostrongyloid nematode Heligmosomoides polygyrus. Responsiveness was also lower among high-ranking (aggressive) males regardless of infection status. Reduced responsiveness in both infected animals and high rankers was associated with elevated serum corticosterone concentration (a potential immunodepressant) and was compounded among high-ranking males by subsequent high aggressiveness. As in previous experiments, only low rankers modulated testosterone secretion in relation to current immunocompetence and corticosterone concentration. The lack of any downregulation of aggression in response to parasite-induced immunodepression contrasted with previous results using antithymocyte serum and may be due to the more localized nature of immunodepression during H. polygyrus infection. However, the additional increase in corticosterone concentration resulting from exposure to female odour and destabilized aggressive social relationships did result in downregulation of aggression among high rankers and of testosterone among mice generally, suggesting that modulation rules of thumb are at least partly dependent on the proximate cues associated with immunodepression.     

The effect of neonatal administration of monosodium glutamate on behavior and blood corticosterone level

DBA/2 male mice were treated with monosodium glutamate (MSG) in a dose of 4 mg/g on 1, 3, 5, 7, 9 days after birth. Saline treated and intact males were used as control groups. MSG treated males displayed decreased number of crossed squares, rearings, entries in the centre and time in the centre of open field in comparison with saline-treated but not intact animals. Time in the light compartment of the light-dark box was increased in MSG-treated mice versus both saline treated and intact animals. MSG administration reduced acoustic startle response but did not affect the magnitude of prepulse inhibition of the startle reflex. Sexual motivation in male mice was reduced by MSG, the same trend was observed after saline treatment. MSG administration increased corticosterone basal level 4-fold while saline treatment did not affect it. These data suggest that neonatal administration of MSG decreases locomotion, exploratory activity, anxiety in male mice, while corticosterone level is increased. Saline treatment increases these parameters (except sexual motivation), and this augmentation is not connected to changes in corticosterone basal level.     

Males of a single-brooded tropical bird species do not show increases in testosterone during social challenges

The challenge hypothesis poses that in socially monogamous vertebrates, males increase circulating testosterone in response to aggressive challenges to promote intense and persistent aggression. However, in bird species that raise only a single brood during short breeding seasons as well as those with essential male parental care, males lack the well-documented testosterone response to social challenges. We tested male behavioral and hormonal responses to social challenges in a neotropical bird species, the buff-breasted wren (Thryothorus leucotis), which is single-brooded with extensive male parental care, but in contrast to most species studied to date, has a long breeding season. We presented live female, male, and paired decoys with song playback for 30 min during pre-breeding and breeding periods. Males responded aggressively to all intruders, but male decoys elicited somewhat weaker responses overall. Responses to female decoys were most intense during pre-breeding, whereas pair decoys elicited stronger responses at breeding. Plasma testosterone concentrations did not differ between challenged and unchallenged males, or among males exposed to different decoys or during different seasons. Plasma corticosterone in pre-breeding males was higher in challenged than unchallenged males and varied positively with the duration of social challenge. Circulating dehydroepiandrosterone concentrations were similar in challenged and unchallenged males, but correlated positively with the proportion of time males spent in close proximity to the decoy. Both testosterone and corticosterone results support recent findings, suggesting that brood number and essential male care, but not breeding-season length, may be important determinants of male hormonal responsiveness during aggressive interactions.     

Changes in masculine sexual behavior, corticosterone and testosterone in response to acute and chronic stress in male rats

Chronic exposure to stressors increases HPA axis activity and concomitantly reduces HPG axis activity. This antagonistic relationship between both these axes has been proposed to underlie the inhibition of reproductive function due to stress. Sexual behavior in males may be the most vulnerable aspect of male reproduction to acute and chronic stress and it has been suggested that alterations in sexual behavior during stress are due to the antagonistic relationship between testosterone and corticosteroids. However, only in a few studies has a correlation between the levels of testosterone and corticosterone, and sexual behavior been made. In this study, we evaluated the effects of different stressors, applied both acute and chronically, on masculine sexual behavior and whether or not these effects on sexual behavior are accompanied by changes in plasma levels of corticosterone and testosterone. Additionally, we evaluated the effect of testosterone treatment on the effects of stress on sexual behavior. Sexually experienced male rats were exposed to one of the following stressors: immobilization (IMB), electric foot shocks (EFS) or immersion in cold water (ICW). Sexual behavior and plasma levels of testosterone and corticosterone were assessed on days 1, 5, 10, 15, and 20 of stress. In a second experiment, males were castrated, treated with 3 different doses of testosterone propionate (TP) and exposed to ICW for 20 consecutive days. Sexual behavior was assessed on days 1, 5, 10, 15, and 20 and steroids were evaluated on day 20. Parameters of masculine sexual behavior were modified depending on the characteristics of each stressor. Mount, intromission and ejaculation latencies increased significantly, the number of mounts increased, and ejaculations decreased significantly in males exposed to EFS and to ICW but not in males exposed to IMB. Associated with these effects, testosterone decreased in the EFS and ICW groups on days 1, 15, and 20. However, corticosterone increased only in males exposed to ICW. In castrated males, TP treatment failed to block the effects of stress by ICW on sexual behavior and corticosterone. These results indicate that the effects of stress on sexual behavior depend on the characteristics of each stressor, and these effects, as well as the decrease in testosterone are not necessarily associated with the increase in corticosterone. The fact that testosterone treatment did not prevent the effects of stress on sexual behavior suggests that other mediators could be involved in the alterations of sexual behavior caused by stress.     

Persistent effects of prenatal, neonatal, or adult treatment with flutamide on the hypothalamic-pituitary-adrenal stress response of adult male rats

To explore the role of androgens in early development on adult hypothalamic-pituitary-adrenal (HPA) function in males, we administered flutamide or vehicle injections: (1) to pregnant dams on embryonic days 15-20; (2) to neonatal pups on days 0-5; or (3) to adults on days 55-60. At approximately 70 days of age, trunk blood was collected to determine corticosterone levels (1) upon removal from the home cage, (2) immediately after 30 min of restraint stress, or (3) 60 min after return to home cage following the stressor. Flutamide treatment resulted in higher basal levels of testosterone and stress levels of corticosterone compared to vehicle treatment, and there was no interaction of treatment with age at time of treatment. This suggests that testosterone is less effective at inhibiting HPA function in flutamide-treated males. In addition, prenatally treated males had higher stress levels of corticosterone than neonatally and adult-treated males, regardless of the type of treatment. There were no differences in CBG levels among the groups. The results suggest that, in males, flutamide treatment has a long-lasting effect on HPA function. These results are consistent with our previous research on neonatally gonadectomized males and the hypothesis of organizational effects of sex hormones on HPA function.     

Sexual experience changes sex hormones but not hypothalamic steroid hormone receptor expression in young and middle-aged male rats

Testosterone is well known to regulate sexual behavior in males, but this is dependent upon prior sexual experience. Aging is associated with decreased libido and changes in testosterone, but the role of experience in these age-related processes has not been systematically studied. We examined effects of age and sexual experience on serum hormones (total testosterone, free testosterone, estradiol, LH) and on numbers of androgen receptor (AR) and estrogen receptor α (ERα) immunoreactive cells in the hypothalamus. Extensive sexual experience was given to male rats at 4 months of age. Rats were euthanized at either 4 months (young) or 12 months (middle-aged (MA)). Comparable sexually naïve male rats were handled and placed into the testing arena but did not receive any sexual experience. Thus, we had four groups: young-naïve, young-experienced, MA-naïve and MA-experienced. Serum hormone levels were assayed, and numbers of AR and ERα cells were quantified stereologically in the medial preoptic nucleus (MPN) and the anteroventral periventricular nucleus (AVPV). Sexually experienced males had significantly elevated serum testosterone and free testosterone in both age groups. Both total and free testosterone were higher, and estradiol lower, in middle-aged than young rats. Experience did not alter either AR or ERα expression in the preoptic brain regions studied. Aging was associated with increased expression of AR, but no change in ERα. These results show that sexual experience can induce short-term and long-term alterations in serum hormones but these effects are not manifested upon their receptors in the hypothalamus.     

The role of testosterone in bib size determination in the male house sparrow Passer domesticus,is age dependent

Secondary sexual signals are thought to indicate individual quality. In order to understand the evolutionary pressures that give rise to such traits it is important to understand the physiological mechanisms underlying their production. The black bib of the house sparrow Passer domesticus is known to function as a badge of social status in males. Past studies have found that the size of the bib in older males is determined, at least partly, by the androgen testosterone. The immunocompetence handicap hypothesis suggests that testosterone has a key role in maintaining honest signalling – it is both involved in the development or expression of sexual signals and is immunosuppressive. In this paper we test experimentally two hypo theses relating to bib size development, whether 1) testosterone is only immunosuppressive in conditions where the natural feedback loop from the testes has been removed, and 2) testosterone is, in addition to influencing the bib size of older males, responsible for the size of the bib in juvenile sparrows. In the first experiment we found that exogenous testosterone administered to intact males during the winter (when LH and FSH levels are very low and were not artificially increased by castration) caused significant immunosuppression, albeit in interaction with the stress hormone corticosterone. Second, we found that exogenous testosterone administration in castrated fledgling male house sparrows had no effect on subsequent post‐juvenile moult bib size relative to controls. Our results suggest that in some circumstances testosterone can be immunosuppressive, but that its role in bib size determination is age‐dependent.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Functional relationships between genotypes and environments in behavior. Effects of different kinds of early social experience on interstrain fighting in male mice

Comparisons are made of the interstrain fighting behavior of adult male mice from two inbred strains (ST-albino; CBA-black-agouti), raised in one of three postweaning social situations: 1) isolated from other males, 2) reared with males of the same strains, or 3) reared with males of the opposite strain. Using the fighting behavior of isolated males as a baseline, it is found that each type of early social experience affects the fighting behavior of the males from each of the two inbred strains differently, indicating that the functional relationships between genotype and environment studied in these experiments are nonadditive. The importance of these results in describing the norm of reaction of a behavioral phenotype and in extrapolating data from one experiment to another is discussed.     

Role of maternal plasma corticosterone elevation in the teratogenicity of secalonic acid D in mice

Secalonic acid D (SAD) is a teratogenic mycotoxin that causes cleft palate in the offspring of treated pregnant mice. To investigate the role of maternal corticosterone in the teratogenicity of SAD, pregnant CD-1 mice were treated with 30 mg/kg of SAD i.p. on day 11 of pregnancy in either 5% (w/v) NaHCO3 or 20% (v/v) dimethyl sulfoxide (DMSO) in NaHCO3. Radioimmunoassay (RIA) was performed to determine plasma corticosterone at 24, 48, 72, and 96 hr after dosing. No interference by EDTA, SAD, DMSO, or pentobarbital was noticed on the RIA. Significant (P less than .01) elevations in plasma corticosterone concentrations were seen 24 and 48 hr following dosing of SAD in NaHCO3 with concentrations reaching a peak just prior to the onset of shelf elevation and fusion. Simultaneous treatment with DMSO, an agent known to antagonize the teratogenic effect of SAD, completely abolished the SAD-induced corticosterone elevation at the 24 hr time point and significantly (P less than .01) reduced it at the 48 hr time point. To evaluate the specificity of the role of corticosterone in the teratogenicity of SAD, plasma samples from mature males similarly treated with either single or multiple doses of SAD ranging from 15 to 45 mg/kg were assayed for corticosterone. A dose of SAD comparable to that used in the pregnant females failed to significantly change plasma corticosterone concentrations in the males. An elevation corresponding only to 75% of that in the females was seen in males receiving multiple doses of SAD totaling three times the dose used in the females. As with females, DMSO completely abolished plasma corticosterone elevation by SAD in the males. These results demonstrate, for the first time, the effect of SAD on a mammalian endocrine system and provide evidence for a specific involvement of elevated maternal plasma corticosterone concentrations in SAD teratogenicity.