Role of postnatal androgens in sexual differentiation of the lordosis-inhibiting effect of central injections of cholecystokinin

The neuropeptide cholecystokinin (CCK) inhibits lordosis behavior when infused into the ventromedial nucleus of the hypothalamus (VMN) of female rats and has no effect when infused into the VMN of male rats. To test whether this sex difference develops under the control of perinatal steroids, male rats were castrated or given sham surgeries within 3 h of birth and female rats were injected with either 0 or 100 micrograms testosterone propionate on postnatal day 5. As adults, these rats were castrated as necessary, implanted with unilateral cannulae directed at the VMN, and tested for their ability to display female sexual behavior and to respond to CCK. Neonatal castration of males prevented defeminization of this response. When treated with 5 micrograms estradiol benzoate (EB), neonatally castrated males showed both lordosis behavior and a profound inhibition of that behavior after infusions of CCK. Neonatally castrated males did not display lordosis behavior when treated with 2 micrograms EB. Control males showed no lordosis behavior and, therefore, no response to CCK. Both doses of EB induced lordosis behavior in neonatally androgenized females. Significantly, these neonatally androgenized females were less responsive to CCK's inhibition of lordosis and were also anovulatory. These results imply that androgens alter the development of CCK responsive circuits as well as defeminize cyclic gonadotropin release. Levels of 125I-sCCK-8 binding in the VMN were correlated closely with an individual's ability to respond to sCCK-8. In summary, the inhibition of female sexual behavior caused by exogenously administered CCK in normal adult female rats appears to be controlled at least partially by levels of CCK receptors in the VMN and to differentiate under the control of perinatally present testosterone.     

Castration, sex steroids, and heterosexual behavior in adult male laboratory-housed stumptailed macaques (Macaca arctoides)

The sociosexual behaviors of six stable male-female pairs of stumptailed monkeys were studied in half-hour pair tests. Their performance before and after castration of the males was compared. The effects of replacement therapy with sex steroids on male-female interaction were studied. Also the effects of new females as sexual partners were investigated. Castration caused a significant decrease in sexual behavior. Individual males could display ejaculatory behavior up to about 1 year postcastration. Dihydrotestosterone propionate (75 mg/week/male) alone or in combination with estradiol benzoate (0.9 or 3 mg/week/male) was not effective in restoring sexual behavior to precastration levels in the three castrated males tested. Replacement therapy with testosterone propionate (75 or 10 mg/week/male) was effective in restoring copulatory behavior in half of the castrated males. In some males the introduction of a new female caused an increase in sexual activity, usually when sexual activity with their familiar partner was low. This occurred both in the castration condition and in the steroid treatment period, suggesting, that low activity was caused by low "motivation" and not by the inability to perform.     

Effect of testosterone and melatonin on social dominance and agonistic behavior in male Tscheskia triton

Social dominance and agonistic behavior play important roles in animal societies. Melatonin and testosterone are closely related to social dominance and agonistic behavior in rodents, but interactions between both of them remain unknown. In this study we investigated the effects of testosterone and melatonin by manipulating photoperiod and castration on social dominance and agonistic behavior in male Tscheskia triton. Castration significantly decreases social dominance of both short- and long-day males, suggesting that testosterone benefits social dominance of males in both breeding and non-breeding seasons. In intact conditions, long-day males tended to dominate short-day males, suggesting that the effect of testosterone on social dominance was a little stronger than melatonin. However, castrated short-day males became dominant over their castrated long-day opponents meaning that high melatonin levels obviously benefit social dominance in males. Hormone implantation indicated that testosterone had no effect on non-breeding condition, but that melatonin was important during the breeding season. Our results indicate that both testosterone and melatonin are important in determining social dominance in male hamsters, and the effect of testosterone appears to be stronger than melatonin. Testosterone is responsible for aggression and social dominance in male hamsters during the breeding season, while melatonin regulates behavior during non-breeding, probably due to the different seasonal secretory patterns of the hormones.     

Perineal muscles and motoneurons are sexually monomorphic in the naked mole-rat (Heterocephalus glaber)

Naked mole-rats are eusocial mammals that live in colonies with a single breeding female and one to three breeding males. All other members of the colony, known as subordinates, are nonreproductive and exhibit few sex differences in behavior or genital anatomy. This raises questions about the degree of sexual differentiation in subordinate naked mole-rats. The striated perineal muscles associated with the phallus [the bulbocavernosus (BC), ischiocavernosus (IC), and levator ani (LA) muscles], and their innervating motoneurons, are sexually dimorphic in all rodents examined to date. We therefore asked whether perineal muscles and motoneurons were also sexually dimorphic in subordinate naked mole-rats. Muscles similar to the LA and IC of other rodents were found in naked mole-rats of both sexes. No clear BC muscle was identified, although a large striated muscle associated with the urethra in male and female naked mole-rats may be homologous to the BC of other rodents. There were no sex differences in the volumes of the LA, IC, or the urethral muscles. Motoneurons innervating the perineal muscles were identified by retrograde labeling with cholera-toxin-conjugated horseradish peroxidase. All perineal motoneurons were found in a single cluster in the ventrolateral lateral horn, in a position similar to that of Onuf's nucleus of carnivores and primates. There was no sex difference in the size or number of motoneurons in Onuf's nucleus of naked mole-rats. Thus, unlike findings in any other mammal, neither the perineal muscles nor the perineal motoneurons appear to be sexually differentiated in subordinate naked mole-rats.     

Gonadal hormones masculinize and defeminize reproductive behaviors during puberty in the male Syrian hamster

Three experiments were conducted to test whether testicular hormones secreted during puberty masculinize and defeminize the expression of adult reproductive behavior. Experiment 1 tested the hypothesis that gonadal hormones during puberty masculinize behavioral responses to testosterone (T) in adulthood. Male hamsters were castrated either before puberty (noTduringP) or after puberty (TduringP). All males were implanted with a 2.5-mg T pellet 6 weeks following castration and tested once for masculine reproductive behavior 7 days after the onset of T replacement. TduringP males displayed significantly more mounts, intromissions, and ejaculations than noTduringP males. Experiment 2 tested the hypothesis that gonadal hormones during puberty defeminize behavioral responses to estrogen (EB) and progesterone (P). Eight weeks following castration, noTduringP and TduringP males were primed with EB and P and tested for lordosis behavior with a stud male. Behavioral responses of males were compared to that of ovariectomized (OVX) and hormone primed females. NoTduringP males and OVX females displayed significantly shorter lordosis latencies than TduringP males. Experiment 3 investigated whether prolonged T treatment or sexual experience could reverse the deficits in masculine behavior caused by the absence of T during puberty. Extending the T treatment from 7 to 17 days did not ameliorate the deficits in masculine behavior caused by absence of T during puberty. Similarly, when the level of sexual experience was increased from one to three tests, the deficits in masculine behavior persisted. These studies demonstrate that gonadal hormones during puberty further masculinize and defeminize neural circuits and behavioral responsiveness to steroid hormones in adulthood.     

Social status and sex independently influence androgen receptor expression in the eusocial naked mole-rat brain

Naked mole-rats (Heterocephalus glaber) are eusocial rodents that live in large subterranean colonies including a single breeding female and 1-3 breeding males; all other members of the colony, known as subordinates, are reproductively suppressed. We recently found that naked mole-rats lack many of the sex differences in the brain and spinal cord commonly found in other rodents. Instead, neural morphology is influenced by breeding status, such that breeders, regardless of sex, have more neurons than subordinates in the ventromedial nucleus of the hypothalamus (VMH), and larger overall volumes of the bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN) and medial amygdala (MeA). To begin to understand how breeding status influences brain morphology, we examined the distribution of androgen receptor (AR) immunoreactivity in gonadally intact breeders and subordinates of both sexes. All animals had AR+ nuclei in many of the same regions positive for AR in other mammals, including the VMH, BST, PVN, MeA, and the ventral portion of the premammillary nucleus (PMv). We also observed diffuse labeling throughout the preoptic area, demonstrating that distribution of the AR protein in presumptive reproductive brain nuclei is well-conserved, even in a species that exhibits remarkably little sexual dimorphism. In contrast to other rodents, however, naked mole-rats lacked AR+ nuclei in the suprachiasmatic nucleus and hippocampus. Males had more AR+ nuclei in the MeA, VMH, and PMv than did females. Surprisingly, breeders had significantly fewer AR+ nuclei than subordinates in all brain regions examined (VMH, BST, PVN, MeA, and PMv). Thus, social status is strongly correlated with AR immunoreactivity in this eusocial species.     

Effects of castration and androgen replacement on male courtship behavior in the red-sided garter snake (Thamnophis sirtalis parietalis)

Following artificial hibernation, sexually mature male garter snakes (Thamnophis sirtalis parietalis) exhibited a decline in courtship behavior irrespective of castration, sham operation, or castration with testosterone replacement therapy. Behavior declined more rapidly in castrated animals with testosterone replacement than in castrated or sham-operated animals. In sham-operated animals, the decline in courtship was accompanied by changes in testicular weight and spermatogenic state from small spermatogenically inactive testes to large spermatogenically active testes. Serum androgen levels were more than fourfold greater in sham-operated animals than in castrated animals; cell height of the androgensensitive renal sex segment was greatest in castrated animals with testosterone replacement and least in castrated animals. These findings indicate that following artificial hibernation, male courtship behavior of T.s. parietalis is independent of the presence of the testes.     

Endocrine control of sexual behavior in sneaker males of the peacock blenny Salaria pavo: effects of castration, aromatase inhibition, testosterone and estradiol

The effects of castration and sex steroid manipulations on the expression of sexual behavior were investigated in a small fish, the peacock blenny, Salaria pavo. In this species, large males defend nests and attract females while small "sneaker" males reproduce by imitating the female morphology and courtship behavior in order to approach nests during spawning events and parasitically fertilize eggs. Sneakers switch into nest holders in their second breeding season, thus displaying both male and female-like sexual behavior during their lifetime. We tested the effects of castration and of an aromatase inhibitor (Fadrozole, F), testosterone (T) or 17beta-estradiol (E(2)) implants on the expression of male and female-like behavior in sneakers. Sneakers were either sham-operated, castrated or castrated and implanted with vehicle, F, T+F or E(2)+F. Seven days after the treatment, sneakers were placed in a tank with a nesting male, two ripe females and an available nest. Castrated fish had lower levels of circulating T and increased the time spent displaying female typical nuptial coloration. T implants had the opposite effect, inhibiting the expression of female-like behavior and coloration. E(2) implants had no significant effect on the display of sexual behavior but the frequency of aggressive displays decreased. The results agree with previous findings in sneakers of S. pavo that demonstrated an inhibition of female-like behavior by 11-ketotestosterone (11-KT). The reported increase in T and 11-KT production when sneakers change into nest holders may thus contribute to behaviorally defeminize sneakers. Contrarily, both T and E(2) failed to promote male-like behavior, suggesting that behavioral masculization during tactic switching depends on other neuroendocrine mechanisms or that the time length of the experiment was insufficient to induce male-like behavioral changes in sneakers.     

Hormonal correlates of dominance in meerkats (Suricata suricatta)

In cooperatively breeding meerkats (Suricata suricatta), individuals typically live in extended family groups in which the dominant male and female are the primary reproductives, while their offspring delay dispersal, seldom breed, and contribute to the care of subsequent litters. Here we investigate hormonal differences between dominants and subordinates by comparing plasma levels of luteinizing hormone (LH), estradiol and cortisol in females, and testosterone and cortisol in males, while controlling for potential confounding factors. In both sexes, hormone levels are correlated with age. In females, levels of sex hormone also vary with body weight and access to unrelated breeding partners in the same group: subordinates in groups containing unrelated males have higher levels of LH and estradiol than those in groups containing related males only. When these effects are controlled, there are no rank-related differences in circulating levels of LH among females or testosterone among males. However, dominant females show higher levels of circulating estradiol than subordinates. Dominant males and females also have significantly higher cortisol levels than subordinates. Hence, we found no evidence that the lower levels of plasma estradiol in subordinate females were associated with high levels of glucocorticoids. These results indicate that future studies need to control for the potentially confounding effects of age, body weight, and access to unrelated breeding partners before concluding that there are fundamental physiological differences between dominant and subordinate group members.     

Time course of androgenic modulation of odor preferences and odor cues in male meadow voles, Microtus pennsylvanicus.

During the breeding season, male meadow voles prefer female over male odors and females prefer male over female odors. Testosterone control of males' odor preferences and production of odors attractive to females differ. A male meadow vole's preference for female versus male odor was still evident 1 week after castration, but not 1 week later. This preference was reinstated in testosterone-treated male voles 2 weeks after the onset of hormone replacement. The attractiveness of male odors to females did not disappear until 3 weeks after castration. The attractiveness of male odors was reinstated 1 week after castrated males were treated with testosterone. The time course for the androgenic modulation of production of odors attractive to females may facilitate breeding. For example, at the end of the breeding season males may emit an odor that is still attractive to females. Similarly, at the beginning of the breeding season males may emit an odor that is attractive to females.     

Castration in Gambel's and Scaled Quail: ornate plumage and dominance persist, but courtship and threat behaviors do not

During the breeding season, testosterone in male birds is often linked to some secondary sexual ornaments, courtship behaviors, and intrasexual aggression. I examined the effect of castration on plumage expression in Gambel's Quail (Callipepla gambelii), a species in which males are highly ornate, and in Scaled Quail (C. squamata), an unornamented species. Using male pairs, each consisting of a castrate and a control, I also assessed whether castration affected (1) the behavior of males, (2) the mating decisions of females, or (3) the outcome of male-male competition. Castration did not alter the plumage of male Gambel's or Scaled Quail. In these species, and some other members of the avian order Galliformes, production of ornate plumage appears to be independent of testosterone. In contrast, castration reduced or eliminated courtship behaviors. Females almost never preferred castrated individuals. During male-male competition, castrates also exhibited lower rates of threat behaviors, which appear to be identical to those used during courtship. Castration did not, however, influence the outcome of male-male competition. Castrates of both species exhibited overt aggression (pecks, chases, displacement) and frequently won male contests. Such results suggest that certain types of aggressive behavior may be testosterone-independent. In both Gambel's and Scaled Quail, male body size correlated positively with dominant individuals.     

Breeding status affects motoneuron number and muscle size in naked mole-rats: recruitment of perineal motoneurons?

Naked mole-rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has one breeding female and one to three breeding males; all other individuals are nonreproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. We recently reported that the striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined to date, are not dimorphic in subordinate naked mole-rats. Here we asked whether sexual differentiation of this neuromuscular system occurs when a subordinate becomes a breeder. The size and number of cells within Onuf's nucleus (homologue of the rat spinal nucleus of the bulbocavernosus) as well as perineal muscle volume were examined in subordinate and breeding naked mole-rats of both sexes. Sex differences in perineal motoneurons were not observed, regardless of social status. To our surprise, however, counts of motoneurons in Onuf's nucleus were increased approximately 30% in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf's nucleus that were characterized by small soma size, and lacked a clear nucleus or nucleolus. Although not exhibiting typical motoneuron morphology, some of these small cells were positive for the motoneuron marker, SMI-32. The neuronal changes correlate with increased perineal muscle volumes in breeders. We propose that small, relatively undifferentiated cells are recruited to the pool of large Onuf's nucleus motoneurons when subordinate naked mole-rats become breeders.     

Some contrasting effects of surgical and “chemical” castration on the behavior of male cynomolgus monkeys (Macaca fascicularis)

In nonhuman primates, surgical castration reduces plasma testosterone levels and male sexual behavior, and testosterone replacement restores them. Chemical castration with compounds that lower plasma testosterone levels is used clinically in the treatment of certain forms of cancer and to reduce aberrant sexual behavior in male sex offenders. In the United States, medroxyprogesterone acetate (MPA) is the drug most used to help reduce serious sexual behavioral problems in men. We were therefore interested in comparing the behavioral effects of MPA treatment (40 mg once a week) in 4 intact male cynomolgus monkeys (4 pairs, 120 tests) with data from an earlier study in our laboratory on 4 males observed before and after surgical castration (16 pairs, 192 tests). Both MPA treatment and surgical castration reduced plasma testosterone to very low levels and decreased ejaculatory activity, but MPA treatment additionally affected measures of male sexual motivation (decreased numbers of male mounting attempts and increased mounting attempt latencies) which were not primarily affected by surgical astration. However, surgical castration decreased intromission ability (percentage of intromitted thrusts per test) and male yawning behavior more rapidly than did MPA treatment. This suggested a hypothesis that different mechanisms could be involved in the behavioral effects—namely, that surgical castration may act primarily via testosterone-dependent peripheral mechanisms, while chemical castration with MPA does so primarily via central mechanisms regulating sexual motivation.     

Sexual orientation, proceptivity, and receptivity in the male rat as a function of neonatal hormonal manipulation

The influence of neonatal androgen on the potential to exhibit feminine sexual behavior was investigated. Male rats castrated on Day 0 but not those castrated on Day 4 or later showed hop/darting, ear wiggling, and lordotic behavior in response to treatment with estrogen and progesterone in adulthood at a frequency equal to that of females. Neonatal treatment with testosterone propionate (1 mg/rat for 4 days) abolished the capacity to show these behaviors. In subsequent experiments, involving castration of male rats at 0 or 4 hr after cesarean delivery, the effect of the postnatal surge of testicular secretions on the expression of female sexual behavior was investigated. No differences were seen in the frequency of hop/darting, ear wiggling, and receptivity between males castrated immediately or 4 hr after delivery. In a preference test where the experimental male could choose between an estrous female and a sexually active male, the neonatally castrated males preferred the company of a male when treated with estrogen and progesterone. The implantation of testosterone resulted in a preference for an estrous female. It was concluded that testicular secretions in the newborn male influence adult sexual orientation and suppress the ability to show proceptive and receptive behaviors.     

Neonatal hormonal influences on the development of proceptive and receptive feminine sexual behavior in rats

The objective of this study was to examine the influence of androgen and of the inhibiting of aromatization of androgen to estrogen during the early neonatal period on the development of receptive (lordosis and acceptance of stimulus male mounting attempts) and proceptive (affiliation with and solicitation of stimulus males) feminine sexual behavior. Within 8 hr of birth, male rats were castrated or received subcutaneous implants of the aromatase inhibitor androst-1,4,6-triene-3, 17-dione (ATD) while females received injections of testosterone propionate (TP). At 90 days of age all treated animals and controls were tested for receptive and proceptive feminine sexual behavior. It was found that androgen present neonatally blocked proceptive as well as receptive behavior patterns in adult rats. The proceptive and receptive feminine sexual behavior patterns displayed by adult males deprived of the effects of androgen neonatally either by castration or by treatment with ATD were comparable to those of normal females.     

Gonadal hormones and sexual behavior in groups of adult talapoin monkeys (Miopithecus talapoin).

Three heterosexual groups of six to eight monkeys were studied; all females were ovariectomized, whereas males were either intact or castrated. Aggressive hierarchies were evident in all groups, with females generally outranking males. When females were treated with estradiol, all males looked more frequently at the latters' sexual skin swellings, but only one male who was both dominant and intact copulated with them. Thus, either castration or low rank resulted in decreased levels of sexual behavior in male talapoins. The sexual behavior of dominant castrated males was restored by testosterone therapy, whereas subordinate castrates never copulated, even after large doses of testosterone, though penile erections and ejaculatory reflex (during masturbation) were restored. Following removal of a dominant male, the sexual behavior of the next male in rank was restored, provided he was not castrated and untreated. In contrast to males, female talapoins showed no consistent correlation between their rank and sexual activity. Estradiol therapy was without overall effect upon the frequency of female mounting behavior, though some females mounted and presented to one another more often. Estradiol treatment also caused females to present to males more frequently, but only to those that were sexually active (i.e., who mounted females).     

Significance of Testosterone in Regulating Hypothalamic Content and In Vitro Release of β-Endorphin and Dynorphin

The effects of castration and testosterone replacement on hypothalamic pools of beta-endorphin and dynorphin and on the basal and corticotropin-releasing factor (CRF)-stimulated release of these peptides from hypothalamic slices in vitro were studied. The experiments were done in adult male rats. The hypothalamic content of both peptides increased significantly within 1 week of castration, and levels remained elevated for up to 4 weeks. Testosterone treatment, begun at the time of castration, prevented these increases. In addition, testosterone replacement 6 weeks after castration reversed peptide levels to normal. Basal in vitro release rates of beta-endorphin and dynorphin were significantly lower from hypothalamic slices derived from 1-week castrated animals than from intact males, and when testosterone was administered in various doses in vivo, basal release rates in vitro increased in a dose-related manner. Hypothalami from rats that had been castrated for 4 weeks, however, showed basal release rates similar to those in tissues from intact controls, a finding indicating that castration initially alters both opioid peptide synthesis and release; later, release is normalized, whereas synthesis remains elevated. CRF was found to stimulate beta-endorphin and dynorphin release from hypothalami from intact and from 1- and 4-week-castrated rats, a result indicating that castration does not alter the response of beta-endorphin and dynorphin neurons to this stimulus.     

Gonadal hormones and conspecific marking in male rats

Urine deposited by a rat on a conspecific was quantified with injections of sodium fluorescein, a substance that changes the color of urine. The hypothesis examined in experiment 1 was that marking the environment and a conspecific would be similarly androgen-sensitive behaviors during each of three stages--before castration, after castration, and with restorative therapy with testosterone propionate. Findings were that castration reduced both forms of marking, and testosterone therapy to castrated males restored environmental marking in a dose-response fashion. However, the findings for social marking were more complex; for example, a physiological 200-micrograms testosterone dosage to castrated males was unable to elevate conspecific marking over the rates of marking by castrates without testosterone replacement. In experiment 2 the ontogeny conspecific marking in juvenile males was examined in relation to the pubertal surge of androgens. Results suggested that juvenile male marking of an adult male decreased despite a pubertal increase of androgens. Conclusions were that testicular hormones influenced both forms of marking but were less important in the social setting. Moreover, conspecific marking is not simply an extension of marking the environment.     

Context-dependent effects of castration and testosterone treatment on song in male European starlings

Most seasonally breeding songbirds display dramatic seasonal fluctuations in plasma testosterone (T) levels and mate attraction behaviors, including song. However, males of some songbird species, such as the European starling (Sturnus vulgaris), continue to sing at high levels after the breeding season, when T levels are basal. In male starlings song during the breeding season functions mainly to attract mates, whereas song during the nonbreeding season appears unrelated to reproduction. This suggests that song produced in a context unrelated to female courtship, unlike song directed toward females, is not regulated by plasma T. In captive males housed in large outdoor aviaries we explored the relationship between plasma T and song produced during the breeding season within and outside a courtship context. This was achieved by determining the effects of castration and subsequent T treatment on song and mate attraction behaviors in both the presence and the absence of a female. Compared to intact males, castrated males did not show reduced song activity in the absence of a female for at least 6 months after the operation, strongly suggesting that the expression of noncourtship song is not regulated by plasma T. Likewise, we found that experimentally elevating T levels in castrated males did not affect noncourtship song rates. However, control castrated males receiving empty implants tended to show reduced noncourtship song rates after implantation. This may have been due to a suppressive effect caused by the presence of the T-implanted castrated males in the same aviary. In contrast, courtship singing was clearly controlled by plasma T: it was abolished by castration and restored by subsequent T replacement when males were housed both individually and in a group situation. High plasma levels of T also appeared necessary for the activation of three other behavioral traits critical for mate attraction, namely, nesthole occupancy, spending time (singing) in a nesthole, and carrying green nesting material into a nesthole.     

Estradiol administration and the sexual activity of castrated male rhesus monkeys (Macaca mulatta)

To examine whether estradiol might be effective in maintaining sexual behavior after castration or after testosterone withdrawal, we have observed male rhesus monkeys during daily 1-hr tests alternately with each of two ovariectomized, estradiol-treated females (four males, four females, eight male-female pairs, 798 tests). Estradiol (2-5 micrograms/kg sc/day) or vehicle was administered in counterbalanced order immediately after castration and again immediately after withdrawal of testosterone propionate treatments (800 micrograms and 1.6 mg sc/day). There were no significant differences in behavior during vehicle and estradiol treatments to indicate that estradiol helped to maintain male sexual activity. Instead, estradiol treatment tended to interfere with the capacity to intromit. This supported the results of other studies, namely, that the systemic administration of estradiol does not enhance the sexual behavior of castrated male macaques, and raises questions about the role of both aromatization and estrogen receptors in the male primate brain.