Independent control of sexual and scent marking behaviors of male gerbils by cells in or near the medial preoptic area

This research studied the role of the medial preoptic area and adjacent cell populations in androgen control of scent marking and sexual behavior in male gerbils (Meriones unguiculatus). Experiment 1 replicated previous research showing that implants of testosterone propionate in or near the medial preoptic area reinstate marking behavior in castrates. Implant sites near the diagonal band of Broca or in the posterior part of the medial preoptic area, near the anterior hypothalamus, are more effective than other sites. Experiment 2 showed that medial preoptic area lesions permanently impair sexual behavior despite testosterone stimulation. Experiments 2–4 showed that lesions in or near the medial preoptic area can also disrupt scent marking; however, this behavior gradually recovered in many lesioned males, especially if they received testosterone. The data suggest that both scent marking and sexual behavior are controlled by androgens acting on cells in or near the medial preoptic area, but the cell populations involved in these two behaviors are probably not the same.     

Social stress decreases marking behavior independently of testosterone in Mongolian gerbils

This study examined the endogenous androgen regulation of the marking behavior in Mongolian gerbils (Meriones unguiculatus). In the first experiment, developmental changes of fecal testosterone levels, ventral gland growth, and the marking frequency of male gerbils were investigated. From 9 weeks of age, marking frequency increased with increases in fecal testosterone levels and ventral gland size. The ventral gland size and marking frequency were significantly correlated to the fecal testosterone level. In the second experiment, we hypothesized that reduction in the marking frequency of subordinate males after social confrontations was controlled by a decrease in the circulating testosterone level, and we followed changes in marking frequency, endocrine status, and ventral gland size after social confrontations in which two adult male gerbils established their social ranks by fighting. As expected, marking frequency and ventral gland size were significantly related to social rank, that is, marking frequency was higher among dominant gerbils and lower among subordinates. In addition, fecal corticosterone levels among subordinates were higher than those of dominant animals. However, neither the fecal and plasma testosterone levels, nor testis size, differed between dominant and subordinate gerbils. These results revealed that endogenous androgen played a role in regulating marking behavior and ventral gland size during the developmental stage and that the reductions in marking frequency and ventral gland size occurring in subordinate males after social confrontations were not directly regulated by androgen changes.     

Hypertrophy of gonadotropin releasing hormone-containing neurons after castration in the teleost, Haplochromis burtoni.

In the African cichlid fish, Haplochromis burtoni, males are either territorial or nonterritorial. Territorial males suppress reproductive function in the nonterritorial males, and have larger gonads and larger gonadotropin-releasing hormone- (GnRH) containing neurons in the preoptic area (POA). We describe an experiment designed to establish the causal relationship between large GnRH neurons and large testes in these males by determining the feedback effects of gonadal sex steroids on the GnRH neurons. Territorial males were either castrated or sham-operated, 4 weeks after which they were sacrificed. Circulating steroid levels were measured, and the GnRH-containing neurons were visualized by staining sagittal sections of the brains with an antibody to salmon GnRH. The soma areas of antibody-stained neurons were measured with a computer-aided imaging system. Completely castrated males had markedly reduced levels of circulating sex steroids [11-ketotestosterone (11KT) and testosterone (T)], as well as 17 beta-estradiol (E2). POA GnRH neurons in castrates showed a significant increase in mean soma size relative to the intact territorial males. Hence, in mature animals, gonadal steroids act as a brake on the growth of GnRH-containing neurons, and gonadal products are not responsible for the large GnRH neurons characteristic of territorial males.     

Hypertrophy of gonadotropin releasing hormone-containing neurons after castration in the teleost,Haplochromis burtoni

In the African cichlid fish, Haplochromis burtoni, males are either territorial or nonterritorial. Territorial males suppress reproductive function in the nonterritorial males, and have larger gonads and larger gonadotropin-releasing hormone- (GnRH) containing neurons in the preoptic area (POA). We describe an experiment designed to establish the causal relationship between large GnRH neurons and large testes in these males by determining the feedback effects of gonadal sex steroids on the GnRH neurons. Territorial males were either castrated or sham-operated, 4 weeks after which they were sacrificed. Circulating steroid levels were measured, and the GnRH-containing neurons were visualized by staining sagittal sections of the brains with an antibody to salmon GnRH. The soma areas of antibody-stained neurons were measured with a computer-aided imaging system. Completely castrated males had markedly reduced levels of circulating sex steroids [11-ketotestosterone (11 KT) and testosterone (T)], as well as 17β-estradiol (E₂). POA GnRH neurons in castrates showed a significant increase in mean soma size relative to the intact territorial males. Hence, in mature animals, gonadal steroids act as a brake on the growth of GnRH-containing neurons, and gonadal products are not responsible for the large GnRH neurons characteristic of territorial males. © 1992 John Wiley & Sons, Inc.     

Correlation between the sexually dimorphic aromatase of the preoptic area and sexual behavior in quail: effects of neonatal manipulations of the hormonal milieu

The aromatase of the preoptic area is significantly more active in males than in females. This sex dimorphism in enzyme activity is still found in birds that have been gonadectomized and treated with a same dose of testosterone. This suggests that the sex difference is not the result of a differential activation by the adult hormonal environment but rather is organized neonatally by steroid hormones. As the central aromatization of testosterone is a limiting step in the activation of copulatory behavior by testosterone, the lower aromatase activity in the preoptic area of females might be responsible, at least in part, for their lower sensitivity to the activating effects of testosterone on behavior. Three experiments were carried out to determine whether early manipulations of the hormonal environment, which are known to differentiate sexual behavior, also affect in a permanent way the aromatase activity in the preoptic area. Injection of estradiol benzoate into male embryos on day 9 of incubation decreased the preoptic aromatase activity in parallel to its demasculinizing effect on behavior. Unexpectedly the same treatment tended to increase enzyme activity in females so that the physiological relevance of the observed enzymatic change remains questionable. In two independent experiments, we confirmed that neonatal ovariectomy of female quail interferes with their behavioral differentiation. Females gonadectomized at 4 days post-hatch showed significantly more male-type sexual behavior as adult in response to testosterone than females gonadectomized at the age of 5 weeks. These experiments also confirmed that the preoptic aromatase activity is higher in males than in females but no evidence for an effect of the age of gonadectomy on the enzyme activity could be obtained. The sex difference and experimental modifications observed in the aromatase activity of the preoptic area were not seen in the posterior hypothalamus demonstrating that these effects are specific. The mechanisms controlling the sex difference in aromatase activity are discussed. The difference might be organized by the action of embryonic steroids as suggested by the changes observed in males injected with estradiol benzoate in egg. Alternatively, activational mechanisms cannot be ruled out at present. In one experiment, the activity of the preoptic aromatase was positively correlated with the sexual activity of the birds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histomorphology of preorbital gland in territorial and non-territorial male blackbuck Antelope cervicapra, a critically endangered species

The preorbital gland is a specialized dermal gland of antelopes which plays an important role in territorial marking behavior and pheromonal communication. To our knowledge, there is little information available on the role of preorbital gland marks in Indian antelopes (blackbucks). Males are seen averting the gland during behavioural display and territorial marking but the functional aspect of this gland has not been examined. Hence, the aim of this study was to describe the histomorphology of the preorbital gland in territorial and non-territorial male blackbucks to determine its morphology and secretory function. The results showed that the preorbital gland is composed of modified sebaceous and apocrine glands. The apocrine gland is lined by simple cuboidal epithelial cells; the serous parts of the secretory products are often seen in the apical portions of the cells. The myoepithelial cells contain actin filaments lying on the basal membranes of the apocrine gland. There are some considerable histological changes in the presence of the sebaceous and apocrine glands in territorial males in comparison to non-territorial males. The following histological changes associated with occurrence of the sebaceous and apocrine glands have been observed in territorial and non-territorial male blackbucks: (1) increase of size of sebaceous and apocrine glands and (2) increase in density of sebaceous and apocrine glands in territorial males compared to non-territorial males. It is suggested that the higher development (i.e., size) and density of sebaceous and apocrine glands in territorial males could depend on hormone production (i.e., testosterone). Based on the histological observation and the role of sebaceous and apocrine glands in the preorbital gland supported by literature, it is possible to conclude that both territorial and non-territorial blackbuck males may produce pheromonal substances through preorbital gland (secretion) for olfactory communication.     

Sexual experience affects reproductive behavior and preoptic androgen receptors in male mice

Reproductive behavior in male rodents is made up of anticipatory and consummatory elements which are regulated in the brain by sensory systems, reward circuits and hormone signaling. Gonadal steroids play a key role in the regulation of male sexual behavior via steroid receptors in the hypothalamus and preoptic area. Typical patterns of male reproductive behavior have been characterized, however these are not fixed but are modulated by adult experience. We assessed the effects of repeated sexual experience on male reproductive behavior of C57BL/6 mice; including measures of olfactory investigation of females, mounting, intromission and ejaculation. The effects of sexual experience on the number of cells expressing either androgen receptor (AR) or estrogen receptor alpha (ERα) in the primary brain nuclei regulating male sexual behavior was also measured. Sexually experienced male mice engaged in less sniffing of females before initiating sexual behavior and exhibited shorter latencies to mount and intromit, increased frequency of intromission, and increased duration of intromission relative to mounting. No changes in numbers of ERα-positive cells were observed, however sexually experienced males had increased numbers of AR-positive cells in the medial preoptic area (MPOA); the primary regulatory nucleus for male sexual behavior. These results indicate that sexual experience results in a qualitative change in male reproductive behavior in mice that is associated with increased testosterone sensitivity in the MPOA and that this nucleus may play a key integrative role in mediating the effects of sexual experience on male behavior.     

Testosterone and sexual experience alter levels of plasma membrane binding sites for progesterone in the male rat brain.

Physiological levels of progesterone act in conjunction with androgens to facilitate copulatory behavior in male rats, mice, and lizards. Radiolabeled progesterone conjugated to bovine serum albumin measured specific binding sites in membrane fractions from male rats that were gonadectomized and testosterone treated, or remained gonadally intact, to determine the role of gonadal steroids on mPR binding. To determine whether behavioral experience could alter binding levels, males either remained sexually na?ve or became sexually experienced. In sexually na?ve males, the highest levels of specific binding occurred in the dorsal portions of the medial preoptic area, with only moderate levels of binding in ventral portions of the medial preoptic area and the dorsal and ventral medial hypothalamus. However, conjugated progesterone binding in these brain regions did not change as a function of testosterone or behavioral manipulations. In contrast, the amygdala responded to behavioral experience with significantly (4-fold) increased binding in gonadectomized, T-treated males with sexual experience. These data indicate that the neuronal plasticity for membrane-associated progesterone binding is regionally specific, being regulated by sexual experience following the reinstatement of testosterone levels, thus suggesting a functional role for plasma membrane activity of progesterone in male rat reproduction.     

Low sex steroids, high steroid receptors: Increasing the sensitivity of the nonreproductive brain

Male aggressive behavior is generally regulated by testosterone (T). In most temperate breeding males, aggressive behavior is only expressed during the reproductive period. At this time circulating T concentrations, brain steroid receptors, and steroid metabolic enzymes are elevated in many species relative to the nonreproductive period. Many tropical birds, however, display aggressive behavior both during the breeding and the nonbreeding season, but plasma levels of T can remain low throughout the year and show little seasonal fluctuation. Studies on the year-round territorial spotted antbird (Hylophylax n. naevioides) suggest that T nevertheless regulates aggressive behavior in both the breeding and nonbreeding season. We hypothesize that to regulate aggressive behaviors during the nonbreeding season, when T is at its minimum, male spotted antbirds increase brain sensitivity to steroids. This can be achieved by locally up-regulating androgen receptors (ARs), estrogen receptors (ERs), or the enzyme aromatase (AROM) that converts T into estradiol. We therefore compared mRNA expression of AR, ERalpha, and AROM in free- living male spotted antbirds across reproductive and nonreproductive seasons in two brain regions known to regulate both reproductive and aggressive behaviors. mRNA expression of ERalpha in the preoptic area and AR in the nucleus taeniae were elevated in male spotted antbirds during the nonbreeding season when circulating T concentrations were low. This unusual seasonal receptor regulation may represent a means for the year-round regulation of vertebrate aggressive behavior via steroids by increasing the brain's sensitivity to sex steroids during the nonbreeding season.     

Hormonal control of sexual and scent marking behaviors of male gerbils in relation to the sexually dimorphic area of the hypothalamus

The sexual and scent marking behaviors of male gerbils are stimulated by testosterone (T) action in the preoptic area (POA) of the hypothalamus. The sexually dimorphic area (SDA) in the posterior POA, which also responds to T, is implicated in this process. This research studied the sensitivities of mating, marking, and the SDA to T metabolites and other steroids. Experiment 1 focused on mating. Male gerbils were implanted at castration with 2-mm Silastic capsules containing T, dihydrotestosterone (DHT), 19-nortestosterone (19-nor T), estradiol (E), or no hormone and were tested 3-7 weeks later. T, E, and 19-nor T maintained intromissions, but E-treated males rarely ejaculated. Controls and DHT-treated males stopped mounting. Experiment 2 compared the ability of these steroids to reinstate marking and mating using the same dose and a larger one (5 mm). Androstenedione, 19-hydroxytestosterone (19-OHT), and E plus DHT were studied as well. Volumes of the SDA and SDA pars compacta (SDApc) were also measured. Only T, 19-nor T, E, and E + DHT reinstated sexual behavior, but all steroids except 19-OHT stimulated marking. E and DHT synergized to elicit mating. For marking, they were no more effective together than alone. Steroid-treated males had larger SDAs than controls. Moreover, steroids that stimulated sexual activity produced larger SDAs than steroids that did not. SDA size correlated with copulatory rate, but not with copulatory efficiency. SDApc size correlated with copulatory efficiency, but not with copulatory rate. Like copulatory rate and efficiency, sizes of the SDA and SDApc did not correlate with each other.     

Sex difference and response to testosterone in gabaergic cells of the medial preoptic nucleus and ventral bed nuclei of the stria terminalis in gerbils

The medial preoptic nucleus (MPN) and ventral bed nuclei of the stria terminalis (BST) are needed to maintain mating in sexually experienced male gerbils and rats. The gerbil ventral BST is also activated with mating, as assessed by Fos expression, as is the medial MPN (MPNm) of both species. In gerbils, many of those mating-activated cells contain glutamic acid decarboxylase (GAD), the enzyme that synthesizes γ-aminobutyric acid (GABA). Some of those cells are projection neurons, but others may release GABA locally. Through actions in the medial preoptic area, GABA inhibits and testosterone (T) promotes male sex behavior. Thus, T may promote mating, in part, by decreasing GAD in MPNm or ventral BST cells. In rats, T increases GAD mRNA in the central MPN (MPNc), where MPN GABAergic cells are densest, but mating behavior does not change in sexually experienced males when the MPNc is ablated. Therefore, this study focused on the MPNm and ventral BST to ask whether their GABAergic cells respond to T or are sexually dimorphic. This was done by visualizing cells immunoreactive (IR) for GAD₆₇, an isoform found primarily in cell bodies, in male and female gerbils and in castrated males with and without T. At both sites, males had more GAD₆₇-IR cells than females, and T decreased GAD₆₇-IR cell numbers in males. Thus, the MPNm and ventral BST have GABAergic cells that are sexually dimorphic and in which T decreases GAD, consistent with local effects of T and GABA on mating.     

Gonadal steroids affect LHRH preoptic cell number in a sex/role changing fish

In diandric sex-reversing fishes, sexually active males and females (primary phase) regularly transform into an alternative reproductive morph, terminal-phase males, that are morphologically and behaviorally distinct. The transformation from primary to terminal phase is associated with a twofold increase in the number of luteinizing hormone-releasing hormone (LHRH) immunopositive cells in the forebrain preoptic area, a region involved in both the initial development and daily control of reproductive physiology and behavior. We now show that implants of 11-ketotestosterone induce increases in LHRH cell number in both primary phase sexes to the level observed in field-collected terminal phase males. Conversely, gonadal steroids had no effect on the number of LHRH preoptic cells in terminal phase males, suggesting that this is indeed a terminal stage in the development of this species. These results demonstrate that transition to the terminal phase by both sexes involves a parallel and convergent change in LHRH cell number, which utilizes an evolutionarily conserved mechanism of sexual differentiation: the inductive effects of gonadal steroid hormones.     

Gonadal steroids affect LHRH preoptic cell number in a sex/role changing fish.

In diandric sex-reversing fishes, sexually active males and females (primary phase) regularly transform into an alternative reproductive morph, terminal-phase males, that are morphologically and behaviorally distinct. The transformation from primary to terminal phase is associated with a twofold increase in the number of luteinizing hormone-releasing hormone (LHRH) immunopositive cells in the forebrain preoptic area, a region involved in both the initial development and daily control of reproductive physiology and behavior. We now show that implants of 11-ketotestosterone induce increases in LHRH cell number in both primary phase sexes to the level observed in field-collected terminal phase males. Conversely, gonadal steroids had no effect on the number of LHRH preoptic cells in terminal phase males, suggesting that this is indeed a terminal stage in the development of this species. These results demonstrate that transition to the terminal phase by both sexes involves a parallel and convergent change in LHRH cell number, which utilizes an evolutionarily conserved mechanism of sexual differentiation: the inductive effects of gonadal steroid hormones.     

Intracranial androgenic and estrogenic stimulation of male-typical behaviors in house mice (Mus domesticus).

Two experiments in house mice (Mus domesticus) examined the neural sites at which steroid hormones activate the following male-typical behaviors: 70 kHz ultrasonic mating vocalizations in response to stimulus females or their urine, urinary marking in response to stimulus males or stimulus females, mounting of estrous females, and intermale aggression. In the first experiment, four groups of castrated males received bilateral intracranial implants of testosterone (T) into either the septum (SEPTUM), medial preoptic area (MPO), anterior hypothalamus (AHA), or ventromedial hypothalamus (VMH). Two control groups received subcutaneous silastic capsules of T (TSIL) or empty silastic capsules (BSIL). The TSIL males performed all behaviors at male-typical levels while the BSIL males were unresponsive. MPO males emitted ultrasonic mating vocalizations at high levels while few vocalizations were seen in males of the other brain implant groups. The VMH, AHA, and MPO males urine marked at higher levels than the BSIL males but did not exhibit the high levels of the TSIL males. Mounting was observed only in MPO and TSIL males. Aggression was rare in males from any of the brain implant groups. In the second experiment, the hormone activity of the implants was increased by using testosterone propionate (TP) or a 50% mixture of estradiol (E2) and cholesterol. The six groups were SEPTUMTP, SEPTUME2, MPOTP, MPOE2, TPSIL, and BSIL. The TPSIL males performed all behaviors at male-typical levels while the BSIL males were unresponsive. TP was effective at restoring vocalizations and urine marking only when placed in the MPO; however, E2 was effective at both sites. Again aggression and mounting were less evident in the brain implanted males. In conclusion, implants of T or TP were effective in restoring ultrasonic mating vocalization when placed into the MPO. MPO implants of T and TP were also effective in stimulating urine marking, although VMH and AHA implants also showed some effectiveness. The restorative effects of E2 were not localized which is probably related to the greater hormonal activity of this treatment. Comparisons of the properties of the various brain implants to restore more than one behavior were discussed.     

Estrogen receptor alpha is required in GABAergic,but not glutamatergic,neurons to masculinize behavior

Masculinization of the altricial rodent brain is driven by estrogen signaling during a perinatal critical period. Genetic deletion of estrogen receptor alpha (Esr1/ERα) results in altered hypothalamic-pituitary-gonadal (HPG) axis signaling and a dramatic reduction of male sexual and territorial behaviors. However, the role of ERα in masculinizing distinct classes of neurons remains unexplored. We deleted ERα in excitatory or inhibitory neurons using either a Vglut2 or Vgat driver and assessed male behaviors. We find that Vglut2-Cre;Esr1^lox/lox mutant males lack ERα in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and posterior ventral portion of the medial amygdala (MePV). These mutants recapitulate the increased serum testosterone levels seen with constitutive ERα deletion, but have none of the behavioral deficits. In contrast, Vgat-Cre;Esr1^lox/lox males with substantial ERα deletion in inhibitory neurons, including those of the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr), posterior dorsal MeA (MePD), and medial preoptic area (MPOA) have normal testosterone levels, but display alterations in mating and territorial behaviors. These mutants also show dysmasculinized expression of androgen receptor (AR) and estrogen receptor beta (Esr2). Our results demonstrate that ERα masculinizes GABAergic neurons that gate the display of male-typical behaviors.     

Parental Investment and Territorial/Sexual Behavior in Male and Female Reed Warblers: Are they Mutually Exclusive?

The behavioral and endocrine changes from prenesting to feeding phases of reproduction were examined in free-living reed warblers. Apart from the onset of feeding behavior, these changes in males are particularly evident in song, territorial behavior and circulating levels of testosterone. The interactions between an individual's physiology and behavior during these behavioral transitions were tested first by correlating song and feeding rates with circulating levels of testosterone and secondly by implanting feeding males with steroids and documenting the resulting effects on song, territoriality and parental behavior. The physiological mechanisms associated with the transition from the sexual phase (nest-building and egg-laying) to the parental (incubation and feeding) in females were also examined. In this case, estrogens were implanted during the parental phase to enhance sexual behavior. The results demonstrate an antagonistic relationship between sexual/territorial behavior and parental care in males which is mediated by changes in circulating androgens and no such antagonism in females. The effects of parental manipulation on the feeding rates of the partners and the growth rates of offspring have been examined to shed light on the necessity for male parental input.     

Activation of sexual behavior by implantation of testosterone propionate and estradiol benzoate into the preoptic area of the male Japanese quail (Coturnix japonica)

Intracranial implantation of minute pellets of gonadal steroids was performed to determine neuroanatomical loci at which steroids activate sexual behavior in the Japanese quail (Coturnix japonica). In this species, systemic treatment of castrated males with either testosterone propionate (TP) or estradiol benzoate (EB) restores male-typical copulatory behavior (head grabbing, mounting, and cloacal contact movements). In addition, EB activates female-typical receptive behavior (crouching). Adult male castrated quail were implanted intracranially with 300-micrograms pellets containing TP, EB, or cholesterol (CHOL) and behavior was tested with intact males and females. Either TP or EB pellets in the preoptic area (POA) activated male-typical copulatory behavior. Mounting was specifically activated without concomitant activation of other steroid-sensitive sexual and courtship behaviors. TP and EB implants in adjacent nuclei containing receptors for these steroids and CHOL implants in POA had no effect on male-typical copulatory behavior. Eighteen percent of all males tested for female-typical receptivity crouched, but no specific effect of EB was seen at any site. The similarity of the POA sites for activation of mounting by TP and EB is consistent with the hypothesis that cells within the POA aromatize testosterone to estrogens, which directly stimulate the cellular processes leading to behavioral activation.     

Experimental induction of social instability during early breeding does not alter testosterone levels in male black redstarts,a socially monogamous songbird

Testosterone plays an important role in territorial behavior of many male vertebrates and the Challenge Hypothesis has been suggested to explain differences in testosterone concentrations between males. For socially monogamous birds, the challenge hypothesis predicts that testosterone should increase during male–male interactions. To test this, simulated territorial intrusion (STI) experiments have been conducted, but only about a third of all bird species investigated so far show the expected increase in testosterone. Previous studies have shown that male black redstarts (Phoenicurus ochruros) do not increase testosterone during STIs or short-term male–male challenges. The aim of this study was to evaluate whether black redstarts modulate testosterone in an experimentally induced longer-term unstable social situation. We created social instability by removing males from their territories and compared the behavior and testosterone concentrations of replacement males and neighbors with those of control areas. Testosterone levels did not differ among replacement males, neighbors and control males. Injections with GnRH resulted in elevation of testosterone in all groups, suggesting that all males were capable of increasing testosterone. We found no difference in the behavioral response to STIs between control and replacement males. Furthermore, there was no difference in testosterone levels between replacement males that had expanded their territory and new-coming males. In combination with prior work these data suggest that testosterone is not modulated by male–male interactions in black redstarts and that testosterone plays only a minor role in territorial behavior. We suggest that territorial behavior in species that are territorial throughout most of their annual life-cycle may be decoupled from testosterone.     

Behavioral phenotypes persist after gonadal steroid manipulation in white-throated sparrows

White-throated sparrows (Zonotrichia albicollis) exhibit a behavioral polymorphism that segregates with a plumage marker. Individuals with a white stripe (WS) on the crown engage in an aggressive strategy that involves more singing, whereas individuals with a tan stripe (TS) sing less and engage in more parental care. Previous work has shown that plasma levels of gonadal steroids differ between the morphs in both sexes, suggesting a hormonal mechanism for the polymorphic behavior in this species. Here, we eliminated morph differences in plasma levels of testosterone (T) in males and estradiol (E2) in females in order to test whether morph differences in behavior would be similarly eliminated. Males and females in non-breeding condition were treated with T or E2, respectively, so that plasma levels in the treated groups were high and equal between the WS and TS morphs. We found that despite hormone treatment, WS and TS birds differed with respect to singing behavior. WS males sang more in response to song playback than did TS males, and WS females exhibited more spontaneous song than TS females. We also found that WS males gave more chip calls, which are often used in contexts of territorial aggression. Overall, these results suggest that WS birds engage in more territorial vocalization, particularly song, than do TS birds, even when T or E2 levels are experimentally equalized. This behavioral difference may therefore be driven by other factors, such as steroid metabolism, receptor expression or function, or steroid-independent neurotransmitter systems.