Hormonal control of precopulatory sebaceous scent marking and ultrasonic mating vocalizations in male rats

The effects of testosterone (T) and its major metabolites, estradiol (E2) and dihydrotestosterone (DHT), on the restoration of sebaceous scent marking and 50-kHz ultrasonic vocalizations in male rats were measured in two studies (Experiments 1 and 2) employing different hormone levels. Silastic capsule administration of high and low doses of T (10 and 20 mm) or E2 (5%; 5 and 10 mm) completely restored marking to precastration levels. Both doses of DHT (30 and 40 mm) or no hormone replacement were without effect when tested in presence of estrous odor cues. In our testing paradigm, males appeared to mark glass objects with a sebaceous secretion rather than urine. Only the high dose of T (20 mm) restored vocalizations to intact levels, while animals receiving the high or low dose of E2 or DHT showed no restoration of behavior. In Experiment 3, habituation to estrous odor cues over weekly tests was not observed for marking or vocalizations. These results are discussed with respect to similarities and differences in the hormonal control of signaling behaviors by androgen metabolites in male rodents.     

Effects of sex hormones and gender on attraction thresholds for volatile anal scent gland odors in ferrets

Body odors contribute to mate recognition and sexual partner preference in many mammals, including ferrets. We used a habituation/dishabituation procedure to test whether sex steroid hormones influence whether ferrets will approach and investigate different concentrations of volatile anal scent gland odors from male and female conspecifics. When tested with high concentrations of anal scent gland secretions in oil vehicle, gonadectomized male and female ferrets that received no sex steroids reliably discriminated anal scents from male and female conspecifics. This discrimination most likely reflects gender recognition rather than individual recognition because gonadectomized, sex steroid-treated ferrets discriminated between anal scents of males and females but not between anal scents of individual males or females. Treatment with either the estrogen receptor agonist, estradiol benzoate (EB), or the androgen receptor agonist, 5-alpha dihydrotestosterone proprionate (DHTP), increased investigation of low concentrations of anal scent by gonadectomized ferrets. These data suggest that ferrets could use anal scent gland secretions in mate recognition and that seasonal increases in circulating sex steroid hormones increase ferrets' responsiveness to low concentrations of these odors.     

Comparing the relative amount of testosterone required to restore sexual arousal, motivation, and performance in male rats

It is well established that male rat reproductive behaviors including sexual arousal, motivation, and performance are dependent on circulating levels of testosterone (T). The present study was designed to (1) compare the relative amount of T required to restore these different aspects of behavior in castrated rats, and (2) create an animal model for clinical populations with sexual impairments. Twenty-nine male Long–Evans rats were tested before and after castration for sexual performance (copulation), motivation (partner preference), and arousal (50 kHz ultrasonic vocalizations; measured together with scent marking). Sexual arousal was also inferred from copulation data. Rats were then assigned to one of four groups, and T was re-introduced via Silastic capsule implants varying in length and content: No T (empty capsules), Low T (2 mm capsules), Medium T (5 mm capsules), or High T (two 10 mm capsules). The highest dose was intended to restore physiological levels. Results indicate that High T is required for 50 kHz vocalizations, while Medium T was sufficient for the restoration of copulation, partner preference, and scent marking. These data suggest that sexual arousal may be most sensitive to reductions in testosterone. The role of T levels in measures of generalized and specific (sexual) arousal is discussed in the context of other reproductive behaviors. Furthermore, because the Low T group showed impairments across all behaviors during post-implant tests, we propose that these animals may provide a good animal model for studying clinical conditions marked by reduced motivation and arousal, including Hypoactive Sexual Desire Disorder.     

Effects of testosterone in the VMN on copulation,partner preference,and vocalizations in male rats

The present study tested whether testosterone propionate (TP) implanted in the ventromedial nucleus (VMN) of the hypothalamus could initiate performance, motivational, or sociosexual components of sexual behavior in castrated male rats. Twenty-seven intact male Long Evans rats were pretested for copulation, partner preference, and 50-kHz vocalization and were subsequently castrated. Approximately 3 weeks after castration, males were retested to confirm that these behaviors had declined, and groups were assigned. Groups 1 and 2 were implanted with bilateral stainless steel cannulae directed at the VMN that were either filled with TP (TVMN group) or remained empty (Blank group). A third group (TSC) was implanted subcutaneously with two 10-mm Silastic capsules filled with testosterone. Restoration of behavior was measured for 2 weeks after implants. We found that copulation and 50-kHz vocalization were not restored by TP in the VMN alone. However, partner preference returned to preoperative levels in both the TVMN and TSC groups, indicating that TP in the VMN was sufficient to restore sexual motivation. Following behavioral testing, prostate glands and seminal vesicles were weighed and confirmed that TP did not leak into the periphery in the TVMN group. Immunostaining for androgen receptors also verified that TP spread was confined to the immediate area surrounding the cannula tip. These results suggest that androgen activation at the VMN is sufficient to induce the motivational components of male sexual behavior, whereas activation of other brain sites is required for copulation and ultrasonic vocalization.     

Long-term effects of pubertal anabolic-androgenic steroid exposure on reproductive and aggressive behaviors in male rats

The current study examined acute and long-term effects of anabolic-androgenic steroid (AAS) exposure during puberty on copulation, vocalizations, scent marking, and intermale aggression, both with and without tail pinch, in intact male rats. Animals received 5 mg/kg of testosterone, nandrolone, stanozolol, or vehicle, beginning at puberty. After 5 weeks, behavior tests were performed while continuing AAS injections. AAS treatment was then discontinued. Behaviors were tested during 3-5 weeks, 9-11 weeks, and 15-17 weeks of withdrawal. During AAS administration, stanozolol males showed significant reductions in all behaviors compared with controls, except aggression with tail pinch. Nandrolone treatment significantly reduced vocalizations and scent marking, and testosterone had no significant effect on behavior. During withdrawal, behaviors in stanozolol males recovered to control levels at variable rates: aggression at 4 weeks; mounts, vocalizations, and scent marking at 9 weeks; and ejaculations at 15 weeks of withdrawal. Stanozolol males showed significantly higher levels of tail pinch-induced aggression during every withdrawal test. Nandrolone-treated males scent-marked at control levels by 9 weeks withdrawal but displayed significantly fewer vocalizations and significantly more tail pinch-induced aggression than controls for the entire study. Testosterone-treated males scent-marked significantly below controls at 3 weeks withdrawal and showed significantly more tail pinch-induced aggression at 5 weeks withdrawal. All three AAS significantly increased tail pinch-induced aggression compared with corresponding nontail pinch tests, even at study endpoint. These results suggest that alterations in androgen-dependent behaviors by pubertal AAS exposure can persist long after drug exposure, and some effects may even be permanent.     

Relationship between 22-kHz calls and testosterone in male rats

Ultrasonic calls in rats induced by the presence of a predator, referred to as “22-kHz calls,” are mainly emitted by socially dominant male rats. Testosterone levels are closely related to social dominance in male rats. In the present study, we investigated the relationship between the emission of stress-induced 22-kHz calls and circulating testosterone levels in male rats, using a combination of surgery (castration or sham operation) and chronic steroid administration (testosterone or cholesterol) to modify circulating testosterone levels. We also assessed the effects of androgen and/or estrogen receptor antagonists on the emission of 22-kHz calls in male rats. An air puff stimulus, known to reliably induce 22-kHz calls in rats, was used as a stressor. Castrated rats with cholesterol implants exhibited significantly fewer 22-kHz calls than rats that had received a sham operation and cholesterol implants, and there was no significant difference between castrated rats with testosterone implants and rats that had received a sham operation and cholesterol implants. Only male rats pretreated with a binary mixture of androgen and estrogen antagonists exhibited significantly fewer 22-kHz calls than controls. These results show that testosterone in male rats has a positive effect on the emission of stress-induced 22-kHz calls, and the calls may be regulated by the activation of both androgen and estrogen receptors.     

Effects of ATD on male sexual behavior and androgen receptor binding: a reexamination of the aromatization hypothesis

The aromatization hypothesis asserts that testosterone (T) must be aromatized to estradiol (E2) to activate copulatory behavior in the male rat. In support of this hypothesis, the aromatization inhibitor, ATD, has been found to suppress male sexual behavior in T-treated rats. In our experiment, we first replicated this finding by peripherally injecting ATD (15 mg/day) or propylene glycol into T-treated (two 10-mm Silastic capsules) or control castrated male rats. In a second experiment, we bilaterally implanted either ATD-filled or blank cannulae into the medial preoptic area (MPOA) of either T-treated or control castrated male rats. With this more local distribution of ATD, a lesser decline in sexual behavior was found, suggesting that other brain areas are involved in the neurohormonal activation of copulatory behavior in the male rat. To determine whether in vivo ATD interacts with androgen or estrogen receptors, we conducted cell nuclear androgen and estrogen receptor binding assays of hypothalamus, preoptic area, amygdala, and septum following treatment with the combinations of systemic T alone. ATD plus T, ATD alone, and blank control. In all four brain areas binding of T to androgen receptors was significantly decreased in the presence of ATD, suggesting that ATD may act both as an androgen receptor blocker and as an aromatization inhibitor. Competitive binding studies indicated that ATD competes in vitro for cytosol androgen receptors, thus substantiating the in vivo antiandrogenic effects of ATD. Cell nuclear estrogen receptor binding was not significantly increased by exposure to T in the physiological range. No agonistic properties of ATD were observed either behaviorally or biochemically. Thus, an alternative explanation for the inhibitory effects of ATD on male sexual behavior is that ATD prevents T from binding to androgen receptors.     

The role of aromatization in androgen stimulation of gerbil scent marking

The androgen dependent scent marking behavior of male Mongolian gerbils (Meriones unguiculatus) can be stimulated after castration by either testosterone or estrogen, but not by dihydrotestosterone (DHT). To determine if DHT fails to evoke scent marking because it cannot be aromatized to form an estrogen, two other nonaromatizable androgens, 1α-methyltestosterone and 6α-fluorotestosterone, were studied. 6α-Fluorotestosterone and its propionate ester stimulated scent marking in castrated male gerbils as effectively as testosterone and its ester did. Hence, an androgen's aromatizability does not determine its ability to influence gerbil scent marking behavior.     

Regulation of sociosexual communication in female Long-Evans rats by ovarian hormones

Two experiments examined the role of the steroid hormones, estradiol (E2), progesterone (P), and testosterone (T), in activating scent marking and 50-kHz ultrasonic vocalizations in ovariectomized Long-Evans rats in response to a devocalized male rat. In Experiment 1, females received, in a counterbalanced order, a series of six hormone treatments consisting of two injections (48-54 and 4 hr before behavioral tests). The six treatments were 8 micrograms E2 followed by 500 micrograms P or oil, 2 micrograms E2 followed by 500 micrograms P or oil, and oil followed by 500 micrograms P or oil. Injections of either the high or low dose of E2 followed by P resulted in high levels of vocalizations. Neither E2 by itself or P by itself were very effective. Surprisingly, none of the hormone treatments were effective in activating marking above the level seen when the females received control injections of oil. Four other hormone treatments were examined in Experiment 2: daily injections of 500 micrograms T, daily injections of 50 micrograms E2, implantation of silastic capsules of E2 (5% E2, 5 mm length) followed by 500-micrograms P injections before behavioral tests, and implantation of silastic capsules of E2 followed by oil injections. Animals receiving E2 by silastic capsule followed by P injection displayed the highest levels of marking and vocalizations across the five weekly tests. These results suggest that while E2 and P synergize for the display of female-typical behaviors similar to the hormonal regulation of lordosis, the mechanism of E2 action may be different for the two signaling behaviors. Scent marking appears to be responsive to the tonic levels of E2 released from silastic capsules.     

Effect of neonatal castration on sex steroid receptor levels in the hypophysis of male rats

The content of estradiol and testosterone cytosolic and nuclear receptors has been studied in the pituitary body of adult male rats gonadectomized on day 1-3 after birth (long-term castrates) or in adulthood (short-term castrates). Intact male rats and long- and short-term castrates had the same level of cytosolic and nuclear estrogen receptors. The number of cytoplasmic and nuclear testosterone-binding sites was identical in the pituitary body of adult intact mice and long-term castrates. Contrastingly, the concentrations of androgen cytosolic and nuclear receptors were significantly lower in neonatally castrated males compared to intact adult animals. The results obtained indicate that nuclear testosterone receptors in the pituitary body mediate negative feedback effect of androgen on the release of luteinizing hormone and that the formation of thin mechanism occurs within the first days of life.     

The effect of social environment on estrogen excretion,scent marking,and sociosexual behavior in tamarins (Saguinus oedipus)

The influence of various social environments on estrogen excretion, scent marking, and the expression of sociosexual behavior was examined in cotton-top tamarins (Sanguinus o. oedipus). Behavioral observations and urine collections were conducted on five females while first housed in their natal family group or in the presence of another cycling adult female and then while housed with an unrelated adult male in a separate cage. Behavioral observations only were conducted on males in natal family groups and while housed with an unrelated adult female. Levels of urinary estrone and estradiol for females were measured by specific radioimmunoassays. Females housed in family groups or in the presence of an adult cycling female showed low and noncyclic patterns of estrogen excretion. Removal from these environments resulted in a rapid increase in urinary estrone and estradiol and three of five females demonstrated cyclic patterns of estrone excretion. Rates of anogenital marking in females were elevated after the social change, and sexual interactions, virtually absent in the original environments, were observed in all five females. In males, similar manipulations of the social environment affected the expression of sexual behavior, but not scent marking. The social environment, therefore has a profound impact on fertility and sociosexual behavior in cotton-top tamarin groups, with implications for callitrichid social structure and behavior.     

Hormonal differentiation of sexual behavior in Japanese quail

The effect of hormones on the development of Japanese quail during the postembryonic period was examined. First, subcutaneous implants of estradiol monobenzoate (EB) and testosterone propionate (TP) were implanted 6–12 hr after hatching. EB and TP had no effect on the differentiation of sexual behavior in genetic males or females. However, EB had marked feminizing effects on plumage in genetic males. Second, the role of gonadal hormones during development was examined by gonadectomizing males and females 6–12 hr after hatching and treating them intramuscularly with EB or TP as adults. EB-treated adult females displayed sexual behavior typical of the genetic female and developed female plumage. A significant proportion of TP-treated females (57%) displayed male sexual behavior patterns. Cloacal gland development and male-type vocalizations were induced. EB-treated males displayed either male or female sexual patterns depending on the stimulus conditions. Third, to test whether bisexuality in gonadectomized males and females is maintained despite steroid treatment and expression of sexual behavior in adulthood, gonadectomized quail which were originally treated with EB received TP and vice versa. The results indicate that in the absence of gonadal hormones after hatching female quail remain bisexual until exposed to estrogen, whereas gonadectomized male quail retain behavioral bisexuality irrespective of prior estrogen or androgen exposure.     

Scent Marking, Sexual Behavior and Aggression in Male Gerbils: Comparative Analysis of Endocrine Control

SYNOPSIS. The aggressive, sexual, and scent marking behaviorsof male gerbils (Meriones unguiculatus) are sensitive to gonadalandrogens, but androgens are not equally important in the controlof each behavior. In this species, territorial residency, prioraggressive experience, and unidentified factors that contributeto large individual differences in aggressiveness, influencethe aggressive behavior of males at least as much as androgensdo. To the extent that androgens affect aggression between malegerbils, they act partially by altering aggressiveness and partiallyby altering production of aggression-eliciting cues. The natureof these cues is unknown. Understanding the role of androgensin aggression in this species is further complicated by theobservation that castration can either increase or decreaseaggression depending on the age at which the surgery is performed.In contrast, androgens play aprimary role in the control ofsexual behavior and scent marking. Both behaviors consistentlydecline following castration despite prior experience of themales. Both behaviors are also controlled by the medial preopticarea-anterior hypothalamus, an area of the brain often implicatedin the control of male sociosexual behaviors. It appears, though,that the sites, and possibly the mechanisms, of hormone actionunderlying scent marking and sexual behavior differ. Studyingboth behaviors in the same species, and whenever possible inthe same individuals, is proving to be a useful technique foridentifying such differences between behaviors as their sensitivityto steroids and to brain lesions.     

Inhibition of serotonin reuptake differentially affects heterosexual behavior of male and female rats

The selective serotonin reuptake inhibitor, Org 6582, inhibits heterosexual copulatory behavior in castrated testosterone-treated male rats. In contrast, Org 6582, and other serotonin reuptake inhibitors (femoxetine and chlorimipramine), facilities lordosis in estrogen- or estrogen + progesterone-primed ovariectomized female rats. This latter effect is not prevented by adrenalectomy. These findings suggest that serotonin is differentially involved in the regulation of heterosexual behavior of male and female rats.     

Steroid regulation of territorial scent marking in the Mongolian gerbil (Meriones unguiculatus)

Mongolian gerbils (Meriones unguiculatus) display a territorial scent marking response associated with a ventral sebaceous gland. In males, both the gland and behavior are androgen dependent and hormone control of marking is delimited to the preoptic area. The present study examines the effects of eleven steroids, injected subcutaneously or implanted into the preoptic area, on territorial marking in adult male castrates. Steroid effects on ventral gland function are also described. The neural target cells that mediate marking respond to a narrower range of steroids than do peripheral target cells in the ventral gland. Testosterone appears to be the only endogenous steroid capable of eliciting marking in males. Other steroids which share molecular commonalities with testosterone (a 17β-hydroxyl group and a double bond involving carbon 4) also induce marking behavior. Central receptors may recognize the relevant endogenous steroid, testosterone, by these features.     

Sociosexual development,pair bond formation,and mechanisms of fertility suppression in female cotton-top tamarins (Saguinus oedipus oedipus)

The effect of various social environments on sociosexual behavior was examined in six young female cotton-top tamarins (Saguinus oedipus oedipus) and in three established breeding females. Behavioral observations and hormonal samples were collected on young females while they were living with their families, when they were isolated from conspecifics, and after they were paired with an unrelated male. While living with the family, all females showed a suppression of fertility and low frequencies of sociosexual behavior. Following removal from the family, isolated females displayed an increase in rate of scent marking and an increase in hormonal levels. When young females were paired with males, they were exposed to scent secretions from their natal families, from an unfamilar family, and from a control for a total of 24 weeks. After pairing, hormonal levels increased dramatically, and ovarian cyclicity began. An increase in sociosexual behavior and elevated levels of scent marking accompanied this physiological change. Newly paired females had higher rates of affiliative behavior and scent marking than did established breeding females. However, both newly paired and established breeding males were more likely to initiate contact, grooming bouts, and social sniffing than were females. Time to first ovulation was later in females who were exposed to scent secretions from their natal families than it was in those females given a control for the first 8 weeks following pairing. No female conceived during exposure to scent secretions. However, once normal ovarian cycling had begun or a pregnancy was established, exposure to scent secretions had no effect. Thus, the social environment influences the fertility, sociosexual behavior, and pair bond formation of cotton-top tamarins. In addition, chemical stimuli found in the scent secretions produced by the natal family are most likely involved in reproductive suppression.     

Androgen- and estrogen-induced copulatory behavior and inhibition of luteinizing hormone (LH) secretion in the male rat

The purpose of the present investigation was to determine if estrogen, aromatizable androgen or nonaromatizable androgen is capable of (1) inducing copulatory behavior and (2) inhibiting the postcastration rise in plasma LH. Castrate male rats were injected daily with either 1 mg testosterone (T), androstenedione (A), dihydrotestosterone (DHT), or 25 μg estradiol benzoate (EB) or oil and tested weekly for masculine behavior and for lordosis behavior after 38 days of steroid treatment. On day 40 blood was collected for radioimmunoassay of plasma LH. At least 89% of the males treated with T, A, or EB and 55% of those treated with DHT displayed ejaculatory behavior whereas none of the oil-treated males showed male copulatory behavior. Only estrogen-treated males displayed lordosis behavior. T and to a lesser extent A treatment reduced high levels of plasma LH; however, DHT and EB further reduced plasma LH to undectable levels. The relative potency of the steroid effect in stimulating accessory sex tissues followed the order: DHT > T > A > EB = oil. Significant dissociation was observed between the effects of these steroids on peripheral morphology, negative feedback, and mating behavior. These results indicate that masculine behavior is facilitated to the greatest extent, although not exclusively, by centrally acting aromatizable androgen or estrogen, whereas under the present conditions only estrogen stimulates feminine behavior.     

Both estrogen receptors and androgen receptors contribute to testosterone-induced changes in the morphology of the medial amygdala and sexual arousal in male rats

In male rats, a steroid-sensitive circuit in the forebrain regulates mating behavior. The masculine phenotype in one component of the circuit, the posterodorsal nucleus of the medial amygdala (MePD), depends on the level of circulating androgens in the adult. To investigate which gonadal steroid receptor(s) mediate sexual arousal and MePD plasticity, adult male rats were castrated and given Silastic capsules containing the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT), 17beta-estradiol (E2), both steroids, or nothing. A fifth group was sham-castrated and treated with blank capsules. DHT treatment was necessary and sufficient to maintain the expression of noncontact penile erections and ultrasonic vocalizations in castrates. E2 had no significant effect on these measures. Both DHT and E2 increased olfactory investigation ("nosepokes") during the noncontact penile erection test. E2, but not DHT, maintained intromission patterns, while either steroid, alone or in combination, maintained ejaculatory behavior. Regional volume and cell soma size of the MePD both decreased following castration. Additionally, MePD cell size was lateralized, with left hemisphere neurons larger than those on the right, an effect that appeared independent of steroid manipulations. DHT and E2 each maintained neuronal soma size. E2 maintained MePD regional volume more effectively in the left MePD than in the right, which may have been due to a greater sensitivity of the left to both castration and hormone treatment. Thus, both androgen receptors and estrogen receptors appear to participate in sexual behaviors that may be mediated by the MePD in adult rats, and both receptors contribute to the steroid-regulated structural plasticity in this brain region.     

Androgen- and estrogen-independent regulation of copulatory behavior following castration in male B6D2F1 mice

Male reproductive behavior is highly dependent upon gonadal steroids. However, between individuals and across species, the role of gonadal steroids in male reproductive behavior is highly variable. In male B6D2F1 hybrid mice, a large proportion (about 30%) of animals demonstrate the persistence of the ejaculatory reflex long after castration. This provides a model to investigate the basis of gonadal steroid-independent male sexual behavior. Here we assessed whether non-gonadal steroids promote mating behavior in castrated mice. Castrated B6D2F1 hybrids that persisted in copulating (persistent copulators) were treated with the androgen receptor blocker, flutamide, and the aromatase enzyme inhibitor, letrozole, for 8 weeks. Other animals were treated with the estrogen receptor blocker, ICI 182,780, via continual intraventricular infusion for 2 weeks. None of these treatments eliminated persistent copulation. A motivational aspect of male sexual behavior, the preference for a receptive female over another male, was also assessed. This preference persisted after long-term castration in persistent copulators, and administration of ICI 182,780 did not influence partner preference. To assess the possibility of elevated sensitivity to sex steroids in brains of persistent copulators, we measured mRNA levels for genes that code for the estrogen receptor-α, androgen receptor, and aromatase enzyme in the medial preoptic area and bed nucleus of the stria terminalis. No differences in mRNA of these genes were noted in brains of persistent versus non-persistent copulators. Taken together our results suggest that non-gonadal androgens and estrogens do not maintain copulatory behavior in B6D2F1 mice which display copulatory behavior after castration.     

Silent or Vocalizing Rats Copulate in a Similar Manner

Both male and female rats produce 50 kHz ultrasonic vocalizations (USVs) in the presence of a sexual partner and during copulation. Previous studies showed that USVs have no incentive value for rats. In this study, we evaluated the role of USVs in behavior during copulation. Three groups of rats were used: sham males paired with sham females, devocalized females paired with sham males, and sham females paired with devocalized males. During the copulation test, the USVs emitted by the sham rat were recorded and the sexual behavior of both the male and the female were observed. The results revealed that devocalized and sham females showed similar patterns of sexual behavior and no difference was found in the copulatory behavior of devocalized and sham males. Also the behavior of the partner of a sham rat was comparable to the partner of a devocalized rat. In addition, almost no changes in USVs emission were found in the 5 seconds before and/or after a copulatory behavior. It can be concluded that USVs play no important role in rat copulatory behavior at least in sexually naïve rats.