Aromatase Inhibitor 1,4,6-androstatriene-3,17-dione Research Articles

Role of membrane estrogen receptor alpha (ERα) in the rapid regulation of male sexual behavior.

The activation of male sexual behavior depends on brain estrogen synthesis. Estrogens act through nuclear and membrane receptors producing effects within hours/days or seconds/minutes, respectively. In mice, estrogen receptor alpha (ERα) is the main estrogen receptor (ER) controlling the activation of male sexual behavior. Although neuroestrogens rapidly modulate mouse sexual behavior, it is not known whether these effects involve membrane ERα (mERα). This study combines two complementary approaches to address this question. C451A-ERα mice carry an ERα that cannot signal at the membrane, while estetrol (E4) is a natural estrogen acting as an agonist on nuclear ERα but as an antagonist on membrane ERα. In wild-type males, E4 decreased the number of mounts and intromissions after 10 min. In C451A-ERα males, E4 also altered sexual performance but after 30 min. E4 did not affect time spent near the female in both wild-type and C451A-ERα mice. However, regardless of genotype, the aromatase inhibitor 1,4,6-Androstatriene-3,17-dione (ATD) decreased both sexual performance and the time spent near the female after 10 and 30 min, confirming the key role of aromatization in the rapid control of sexual behavior and motivation. In conclusion, the shift in timing at which the effect of E4 is observed in mice lacking mERα suggests a role for mERα in the regulation of rapid effects of neuroestrogens on sexual performance, thus providing the first demonstration that E4 acts as an antagonist of a mER in the brain. The persisting effect of ATD on behavior in C451A-ERα mice also suggests the implication of another ER.

Hippocampal Aromatization Modulates Spatial Memory and Characteristics of the Synaptic Membrane in the Male Zebra Finch.

The estrogen-synthesizing enzyme aromatase is abundant at the synapse in the zebra finch hippocampus (HP), and its inhibition impairs spatial memory function. To more fully test the role of local estradiol (E2) synthesis in memory, the HP of adult male zebra finches was exposed to either control pellets or those containing the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), ATD and E2, ATD and the G protein-coupled estrogen receptor (GPER) agonist G1, or the antagonist G15 alone. Birds were tested for spatial memory acquisition and performance, and HP levels of the postsynaptic protein PSD95 were measured. ATD-treated birds took longer to reach criterion than control birds, whereas acquisition in ATD+E2 and ATD+G1 birds was indistinguishable from control and ATD treatments. Interestingly, all G15 birds failed to acquire the task. Following a retention interval, ATD birds took the longest to reach the (formerly) baited cup and made the most mistakes. ATD+E2 animals displayed the lowest retention latencies and made fewer mistakes than ATD-treated birds, and ATD+G1 birds did not significantly differ from controls in retention latencies. The amount of PSD95 in the HP was lowest in ATD-treated animals compared with birds with silicone-only-implanted craniotomies, ATD+E2, and ATD+G1 birds, who did not differ in this expression. Thus, spatial memory acquisition and performance appear aromatase and E2 dependent, an effect more reliably revealed after consolidation and/or recall compared to acquisition. E2 may exert this effect via GPERs, resulting in an increase in PSD95 levels that may modify receptor activity or intracellular signaling pathways to increase synaptic strength.

Prenatal administration of letrozole reduces SDN and SCN volume and cell number independent of partner preference in the male rat

During development, the exposure to testosterone, and its conversion to estradiol by an enzyme complex termed aromatase, appears to be essential in adult male rats for the expression of typical male sexual behavior and female-sex preference. Some hypothalamic areas are the supposed neural bases of sexual preference/orientation; for example, male-oriented rams have a reduced volume of the sexually dimorphic nucleus (oSDN), while in homosexual men this nucleus does not differ from that of heterosexual men. In contrast, homosexual men showed a larger number of vasopressinergic cells in the suprachiasmatic nucleus (SCN). Interestingly, male rats perinatally treated with an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), also showed bisexual preference and an increased number of vasopressinergic neurons in the SCN. However, this steroidal aromatase inhibitor has affinity for all three steroid receptors. Recently, we reported that the prenatal administration of the selective aromatase inhibitor, letrozole, produced a subpopulation of males with same-sex preference. The aim of this study was to compare the volume and number of cells of the SDN and SCN (the latter nucleus was immunohistochemically stained for vasopressin) between males treated with letrozole with same-sex preference, males treated with letrozole with female preference and control males with female preference. Results showed that all males prenatally treated with letrozole have a reduced volume and estimated cell number in the SDN and SCN, independent of their partner preference. These results indicate that the changes in these brain areas are not related to sexual preference, but rather to the effects of letrozole. The divergent results may be explained by species differences as well as by the critical windows during which the aromatase inhibitor was administered.

Postnatal Aromatase Blockade Increases c-fos mRNA Responses to Acute Restraint Stress in Adult Male Rats

Recent evidence suggests that the aromatization of testosterone to estrogen is important for the organizing effects of neonatal testosterone on neuroendocrine responses to acute challenges. However, the extent to which neonatal inhibition of aromatase alters the stress-induced activation of neural pathways has not been examined. Here we assessed central patterns of c-fos mRNA induced by 30 min of restraint in 65-d-old adult male rats that were implanted with sc capsules of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12 h of birth and removed on d 21 of weaning. Neonatal ATD decreased the expression of arginine vasopressin within extrahypothalamic regions in adults, confirming reduced estrogen exposure during development. As adults, ATD-treated animals showed higher corticosterone responses at 30 min of restraint exposure compared with control animals as well as higher c-fos expression levels in the paraventricular nucleus of the hypothalamus. ATD treatment also increased stress-induced c-fos within several limbic regions of the forebrain, in addition to areas involved in somatosensory processing. Based on these results, we propose that the conversion of testosterone to estrogen during the neonatal period exerts marked, system-wide effects to organize adult neuroendocrine responses to homeostatic threat.

Postnatal blockade of androgen receptors or aromatase impair the expression of stress hypothalamic-pituitary-adrenal axis habituation in adult male rats

Sex steroid hormones during development permanently alter, or organize, the brain and behavior, while during adulthood they act to reversibly modulate, or activate, physiology and behavior. Testosterone exerts both organizational and activational effects on the magnitude of the hypothalamic-pituitary-adrenal (HPA) axis response to acute stress. What has never been approached is how testosterone can organize habituation of the HPA axis, in which stress induced elevations in ACTH and corticosterone release decline over repeated exposures to the same stimulus. In the current study we examined HPA responses to repeated psychogenic stress in 65-day-old, adult male rats that received subcutaneous capsules containing the antiandrogen flutamide or the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12h of birth and removed on day 21 of weaning. An additional group of castrated, adult male rats were used to differentiate organizational from activational effects of testosterone. All treatment groups displayed smaller declines in ACTH in response to repeated restraint compared to control animals. Remarkably, the normal decline in corticosterone failed to occur in flutamide- and ATD-treated animals. By contrast, males that were castrated as adults showed a significant reduction in corticosterone after repeated stress. Taken together, these findings underscore an organizing influence of both androgen receptors and estrogen conversion on HPA habituation to repeated psychogenic stress, which appears to occur independent of the activational effects of testosterone.

Sex steroids are necessary in the second postnatal week for the expression of male alloparental behavior in prairie voles (Microtus ochragaster).

Sex steroids play a significant role in organizing male social behavior, which is associated with low levels of pro-social behavior and high levels of aggression. However, the role of steroids in organizing behavior in highly social males is unclear. The authors tested the hypothesis that low levels of sex steroids facilitate the expression of pro-social behavior in male prairie voles (Microtus ochragaster), predicting that inhibition of testosterone and estradiol would reduce spontaneous-alloparental behavior. Treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or the androgen receptor blocker flutamide, days 8-14, significantly reduced the expression of alloparental behavior in 21-day old males. While both treatments reduced alloparental behavior and increased pup-directed aggression, there were differential treatment effects. Flutamide altered initial response, increasing latency to enter the pup cage and the likelihood of retreat from initial contact. ATD-treated males that were alloparental showed increases in sniffing and latencies to lick and huddle. Results indicate that endogenous steroids play a role in the development of male pro-social behavior and the effects of estrogens and androgens differ.

The aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) reduces disinhibitory behavior in intact adult male rats treated with a high dose of testosterone

Anabolic androgenic steroids and high testosterone doses have been reported to induce impulsive behavior in man and behavioral disinhibition in rats. The purpose of the present study was to investigate whether aromatization of testosterone to estradiol is of importance for the behavioral disinhibiting effect of a high testosterone dose in adult male rats. Testosterone administered via five testosterone-filled silastic capsules implanted subcutaneously (s.c.) to non-castrated, group-housed rats for six days induced behavioral disinhibition in a modified Vogel's drinking conflict model and yielded supraphysiological serum levels of testosterone and increased accessory sex organ weights. Moreover, concurrent administration of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD; 60mg/kg/day s.c.) decreased behavioral disinhibition in testosterone-treated rats (without affecting accessory sex organ weights) while behavior was not significantly affected in sham-treated animals. Since some reports indicate that ATD, in addition to inhibit aromatase, also may affect the binding of testosterone to the androgen receptor, the effect of the non-steroidal androgen receptor antagonist flutamide was investigated. Flutamide treatment did not affect disinhibited behavior in testosterone-treated rats. However, in sham-treated animals, flutamide (50mg/kg/day) produced behavioral disinhibition. These results suggest that estradiol is of importance in the mechanisms underlying behavioral disinhibition in non-castrated rats treated with a high testosterone dose. Speculatively, aromatization may be involved in pro-impulsive effects of high testosterone doses in humans.

Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles ( Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.

Endogenous Oestradiol Regulates Progesterone Receptor Expression in the Brain of Female Rat Fetuses: What is the Source of Oestradiol?

Testosterone secreted by male testes during fetal development is aromatized to oestradiol (E(2)) or reduced to the androgen, dihydrotestosteorne (DHT), within specific tissues. The female brain is assumed to develop in the relative absence of gonadal steroid hormones, as the ovary is steroidogenically quiescent until later in postnatal life. However, the proximity of a female fetus to male littermates in utero can increase her exposure to testosterone, and thereby its metabolites. To date, it is has been difficult to dissociate the effects of male-derived E(2) from those of DHT on the developing female brain. In the present study, anogential distance (AGD) in females was used as an androgen-dependent bioassay, whereas progesterone receptor (PR) expression within the medial preoptic nucleus (MPN) was used as an E-dependent measure. Pregnant dams received the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), or vehicle from embryonic day 16 (ED16) to ED21. On ED22, AGD and PR-immunoreactivity (-ir) were measured in females that had zero, one, or two males (0-2M) or females that had three, four, or five males (3-5M) in the uterine horn. AGD was significantly greater in 3-5M females compared to 0-2M females, suggesting that male littermates are the source of androgenic exposure in the female fetus. ATD treatment significantly decreased PR-ir in the MPN, demonstrating E(2) regulation of PR. However, the total number of males in the uterine horn did not effect PR expression. There was no correlation between PR-ir and AGD, suggesting that these measures are influenced independently. Together, these results suggest that although male littermates provide a significant source of androgens to female fetuses, the amount of E(2) aromatized from male-derived testosterone may not be the only biologically relevant source of androgens or E(2). Alternative sources of E(2) may be essential in ensuring the normal development of the female brain.

The role of sex steroids in courtship, pairing and pairing behaviors in the socially monogamous zebra finch

The purpose of this study was to test whether sex steroid actions are necessary for courtship and pairing in socially monogamous birds. We examined the effects of an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), combined with an anti-androgen, flutamide (F), on the behavior and pairing status of initially unpaired male and female zebra finches ( Taeniopygia guttata). In the first experiment, 24 adult males were implanted with either a combination of ATD and flutamide or empty implants. Two weeks after implantation, birds were housed in aviaries containing 3 ATD + F males, 3 control males, and 3 females and allowed 2 weeks to pair, with observations 7 times during the 2-week period. A second experiment tested the effects of these same treatments in females. During the first 4 days of testing, ATD + F males were less likely to attack conspecifics than were control males. ATD + F males were also less likely to “greet,” or approach, females than were control males, but other courtship behaviors, including directed singing, were unaffected. ATD + F females did not differ from control females on any courtship behavior measured. Furthermore, these treatments did not affect pairing behaviors (time spent clumping or in a nest box together) or the likelihood of pairing with a partner of the opposite sex. ATD + F treatments in females did, however, increase the likelihood of same-sex pairing. This suggests that, although sex steroids may regulate some courtship behaviors in males, they do not regulate pairing behaviors and have little effect on the likelihood that a male or female will be chosen as a mate by a bird of the opposite sex.

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Effect of prenatal androgen receptor antagonist or aromatase inhibitor on sexual behavior, partner preference and neuronal Fos responses to estrous female odors in the rat accessory olfactory system

Many socially relevant odors are detected in rodent species by the vomeronasal organ and subsequently processed by the accessory olfactory system (AOS). We previously found that gonadectomized male and female rats treated in adulthood with testosterone propionate (TP) showed equivalent Fos responses in the AOS to odors derived from estrous females. Likewise, in contrast with numerous other mammalian species, gonadectomized female rats show surprisingly high levels of male-typical mounting behavior in response to adult TP. We tested the hypothesis that prenatal testosterone (T) exposure, acting via androgen receptors (ARs) or via estrogen receptors, masculinizes the AOS in rats of both sexes. Pregnant dams were treated with either the AR blocker, Flutamide, the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), or nothing (control) to assess the role of prenatal androgen and estradiol receptor activation, respectively, in this masculinization. Beginning at birth, male and female offspring were injected subcutaneously (sc) every other day with either ATD (pre- and neonatal ATD group) or oil vehicle (Flutamide and control groups) until postnatal Day 12. Subjects were gonadectomized as adults, hormonally treated and tested for different behaviors before having their AOS Fos responses to estrous female odors assessed. Prenatal treatment with Flutamide (but not ATD) significantly decreased anogenital distance and severely impaired intromissive and ejaculatory behaviors in males tested after TP replacement without disrupting mounting capacity in either sex. Pre- and neonatal treatment with ATD (but not Flutamide) enhanced lordosis responsiveness in males tested after sc injections of estradiol and progesterone, whereas these perinatal treatments had no effect on any aspect of masculine coital performance in either sex. After TP treatment, male and female control subjects preferred to approach a tethered stimulus female as opposed to a male, and prenatal Flutamide or perinatal ATD treatments did not modify this pattern of partner preference. Neuronal Fos responses to estrous odors were (as in previous studies) identical in the AOS of gonadectomized TP-treated control males and females. Prenatal Flutamide or perinatal ATD treatments failed to disrupt consistently this profile of Fos responses to estrous odors in the AOS of rats of either sex. These behavioral and neuroanatomical findings raise the possibility that the similar level of male-typical responsiveness to social odors that occurs in male and female rats after adult TP treatment results from nonsteroid-hormone-dependent, species-specific factors that act perinatally in the brains of rats of both sexes.

Feedback Control of Gonadotropins in Atlantic Salmon,Salmo salar,Male Parr: II. Aromatase Inhibitor and Androgen Effects

Both positive and negative feedback on the (hypothalmus)– pituitary–gonad axis occur in salmonids. The aim of the present study was to investigate the role of different androgens, and in particular the involvement of aromatization of androgens to estrogens in feedback mechanisms. Previously mature Atlantic salmon,Salmo salar,male parr were studied in two experiments. In the first experiment, intact fish were implanted with Silastic capsules filled with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in spring and sampled in the autumn when the rematuring males were starting to display running milt. In the second experiment, castrated males were implanted with capsules containing ATD, the androgens testosterone (T) and 11-ketoandrostenedione (11KA), or ATD and T combined in spring. These fish were sampled in the summer when rematuring fish were starting to show signs of gonadal growth. Pituitary and plasma gonadotropins (GTH I and GTH II) were studied using radioimmunoassay. In autumn, ATD treatment reduced pituitary and plasma GTH II levels. In summer, GTH II was consistently nondetectable in plasma. Castration diminished pituitary GTH II content. Treatment with T increased pituitary GTH II content, an effect that was attenuated when T treatment was combined with ATD. All these results are consistent with the presence of an aromatase-dependent positive feedback of T on GTH II. 11KA also had a stimulatory effect on GTH II, although weaker than that of T. Testicular size and spermiation was reduced by ATD in autumn; the latter of these results is likely to be due to the inhibitory effect of ATD on GTH II. Positive effects of ATD on plasma GTH I were found in autumn, indicative of an aromatase-dependent negative feedback in this phase. On the other hand, castration increased plasma and pituitary GTH I levels in summer, indicating that the gonads in this phase exert a predominantely negative control of GTH I. In summer, negative effects of T on GTH I pituitary levels were not suppressed, but were rather enhanced, by the combined treatment with ATD. Furthermore, plasma GTH I levels were lower after treatment with T in combination with ATD than with T or ATD separately. These negative effects of T were not diminished by ATD, so that they are nonaromatase-dependent. Furthermore, since they were actually more pronounced in the presence of ATD it is suggested that there is also a positive aromatase-dependent feedback component in this phase. In addition, 11KA had a negative effect on plasma and pituitary GTH I in castrated previously mature males. Thus, GTH I secretion is controlled by both aromatase-dependent and nonaromatase-dependent feedback effects, of which at least the former may be positive or negative depending on season. In summary, the feedback control of GTH I appears to be more complex than that of GTH II.

Flesinoxan: a prosexual drug for male rats

Two tests were carried out to compare the stimulatory (i.e., prosexual) effects of the 5-HT 1A receptor agonists flesinoxan and 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) on sexual behavior in male Wistar rats. Two groups of rats were used: normal males and males with impaired masculine sexual behavior due to neonatal treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). In Experiment 1, flesinoxan (0.3 and 1.0 mg/kg) stimulated ejaculation frequency and number of animals displaying this behavior, both in controls and ATD males. With 0.3 mg/kg flesinoxan ATD males did not differ from controls in ejaculation frequencies. There was a concomitant decrease in latency to first ejaculation. No `premature' ejaculations (i.e., at first or second intromission) were observed. In Experiment 2, the effects of 0.4 mg/kg 8-OH-DPAT, 0.3, 1.0 and 3.0 mg/kg flesinoxan and saline were tested in two ejaculation series. `Premature' ejaculations only occurred during first ejaculation series with 8-OH-DPAT in 8 of 10 controls and in 6 of 9 ATD males; it did not occur during flesinoxan treatment nor in the second ejaculation series with 8-OH-DPAT treatment. Thus, flesinoxan stimulates sexual behavior in control rats and in rats with impaired sexual behavior. Unlike 8-OH-DPAT flesinoxan does not render them into `premature' ejaculators. Therefore, flesinoxan could be considered a prosexual drug for male rats.

NEONATAL INHIBITION OF BRAIN ESTROGEN SYNTHESIS ALTERS ADULT NEURAL FOS RESPONSES TO MATING AND PHEROMONAL STIMULATION IN THE MALE RAT

Neonatal inhibition of brain estrogen formation in male rats by administration of the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), permanently changes aspects of their mating behavior and partner preference in adulthood. The medial preoptic area receives chemosensory inputs via a sexually dimorphic vomeronasal projection circuit, which responds to reproductively relevant pheromonal cues. The medial preoptic area also receives genital somatosensory inputs via the midbrain central tegmental field and the medial amygdala. We used Fos immunoreactivity as a marker of neuronal activation to determine whether there is a correspondence between the behavioral profiles of neonatally ATD-treated male rats and their neuronal responses in the medial preoptic area and other brain regions to somatosensory and chemosensory stimuli.Achieving eight intromissions with an estrous female led to a greater neuronal Fos immunoreactivity in the medial preoptic area of neonatally ATD-treated male rats compared with neonatally cholesterol-treated male rats. Exposure for 1.5 h to chemosensory cues derived from soiled bedding of estrous females induced Fos immunoreactivity throughout the vomeronasal pathway (i.e. medial amygdala, bed nucleus of the stria terminalis and medial preoptic area) in both ATD and cholesterol males (Experiment 2a). By contrast, exposure for 1.5 h to chemosensory cues derived from soiled bedding of sexually active males revealed clear differences between ATD and cholesterol males in neuronal Fos immunoreactive (Experiment 2b). At peripheral portions of the vomeronasal pathway (i.e. the accessory olfactory bulb and the medial amygdala), there were no differences in the number of Fos-immunoreactivity neurons between ATD and cholesterol males. However, neurons in the more central portions of the vomeronasal pathway (i.e. the bed nucleus of the stria terminalis and the medial preoptic area) showed increased Fos immunoreactivity after exposure to odors from sexually active males in ATD males as opposed to cholesterol males. Females, like ATD males, showed neuronal Fos immunoreactivity at each level of the vomeronasal pathway after being exposed to odors from sexually active males.These results suggest that the responsiveness of neurons in the central portion of the vomeronasal projection circuit to odors from sexually active males, but not estrous females, is sexually differentiated in male rats due to the neonatal action of estrogens. Copyright © 1996 IBRO. Published by Elsevier Science Ltd.

Sexual differentiation of odor and partner preference in the rat

Previous studies have shown that adult male rats, in which brain estrogen formation was inhibited neonatally by SC administration of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), show an altered sexual partner preference. When tested in a three-compartment box, such gonadally intact ATD males approach and mate both with the estrous female and the sexually active male, whereas normal males prefer to approach and mate with the estrous female, avoiding the stimulus male. After castration in adulthood and estradiol treatment, ATD males prefer sexually active males. Similarly treated normal males prefer estrous females, and estrous females prefer to mate with males. In the present study, we asked what stimulus characteristics of active males vs. estrous females determined the different sexual preferences of males, ATD males, and of females. Were they chemosensory cues or more distal cues such as actually seeing and hearing the stimulus animals or the reward of sexual activity with the stimulus animals? Sex differences in preference were evident when animals were given a choice between soiled bedding from estrous females and from sexually active males. ATD and control males spent significantly more time on soiled bedding from estrous females than on soiled bedding from sexually active males. Control females spent significantly more time on soiled bedding from sexually active males than on soiled bedding from estrous females. More distal cues, such as seeing and hearing the stimulus animals, revealed differences in preference between control males and females, but not between ATD and control males. Physical interaction with the stimulus animals was a prerequisite for revealing differences in preference between ATD and control males. Then, the behavior of ATD males was clearly intermediate between that of normal male and female rats. In conclusion, neonatal estradiol is important for the psychosexual development of the male rat. However, the present data suggest that the psychosexual development of the male rat also requires either prenatal estradiol or perinatal testosterone.

Endogenous reproductive hormones and nocturnal rhythms in partner preference and sexual behavior of ATD-treated male rats.

Male rats received subcutaneously silastic capsules, containing the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), shortly after birth. Control males were given silastic capsules containing cholesterol. The capsules were removed at the age of 21 days. In adulthood, blood serum was collected early and late in the dark phase of the light/dark cycle (experiment I). Testosterone and luteinizing hormone and follicle stimulating hormone (FSH) fluctuated nocturnally, both in ATD and control males, with highest levels late in the dark phase. FSH levels were significantly higher in ATD males. Nocturnal levels of inhibin, a selective suppressor of pituitary FSH secretion, also fluctuated in both ATD and control males, with lowest levels late in the dark phase. In experiment II, ATD and control males were tested for partner preference behavior in a three-compartment box (choice: sexually active male vs. estrous female) early and late in the dark phase. When gonadally intact, ATD males, but not controls, showed a clear nocturnal rhythmicity in partner preference behavior and sexual behavior. Early in the dark phase, such ATD males preferred the vicinity of and interaction with a sexually active male. Late in the dark phase, this preference for the active male shifted to a preference for the estrous female. Control males preferred the estrous female. After castration and subsequent treatment with testosterone via silastic capsules, which ensured constant blood serum levels, ATD males continued to show their nocturnal rhythms in partner preference behavior and in sexual behavior. Thus, the underlying mechanism of the nocturnal rhythmicity phenomenon is an organizational effect of neonatal ATD treatment rather than an activational effect of fluctuating serum hormone levels.

Differential effects of aromatase inhibition on luteinizing hormone secretion in intact and castrated male cynomolgus macaques.

To understand the role of central aromatization in feedback regulation of LH in nonhuman primates, we treated adult male cynomolgus monkeys with the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD). We measured LH, testosterone (T), and ATD in systemic sera of blood samples drawn on a diurnal schedule (0900 and 2100 h). Each animal was bled for 4 pretreatment days from a femoral catheter after which they were divided into the following treatment groups: castrated (Cx), n = 2; Cx + T, n = 6; Cx + T + ATD, n = 6; Cx + ATD, n = 3; and sham operated + ATD, n = 3. Silastic capsules or packets containing T or ATD, respectively, were placed sc between the scapulae at the time of Cx or sham treatment. In T-treated animals, T (20 micrograms/kg body weight) dissolved in propylene glycol was injected im at 2100 h to mimic the diurnal rise of T observed in nonhuman primates. Animals were bled for 2 weeks after which they were killed, and selected brain areas were analyzed for aromatase activity and cytosolic and nuclear androgen receptors. Animals treated with ATD had significantly reduced levels of aromatase activity in selected regions of the hypothalamus, preoptic area, and the amygdala (P < 0.05). Even though ATD inhibited brain aromatase activity, it did not prevent the negative feedback actions of T on LH secretion after Cx. In addition, ATD by itself inhibited LH secretion after Cx and activated brain androgen receptors. These latter effects of ATD seemed to have been mediated through a metabolite. In sham-operated intact males, ATD produced variable surges of LH that were accompanied by elevations of T in the systemic circulation. These differential effects of ATD in intact vs. castrated animals demonstrate the importance of selecting the proper model system to study LH control mechanisms. In the intact animal, aromatization seems to play a role in regulating LH secretion, but the postcastration rise of LH seems to be regulated differently.

Aromatase inhibitors regenerate the thymus in aging male rats

The thymus can be regenerated in aging rats by surgical or chemical castration and regeneration is inhibited by testosterone, which may exert this effect, at least in part, through its conversion to estradiol. An attempt has been made to regenerate the thymus in intact aging rats using inhibitors of the aromatase system, in the hope that this maneuver could lead to the use of such chemical intervention in the treatment of immunodeficiency syndromes. Young adult and aging (18-month-old) male rats were orchidectomized under ether anesthesia and 7 days later given s.c. implants of testosterone in silicone elastomer (SILASTIC) tubing. Some rats received testosterone together with a five-fold excess of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). One group of young intact rats received implants containing 25 mg ATD and group of 18-month-old intact rats received 125 mg ATD or 25 mg of another, more powerful aromatase inhibitor 4-hydroxyandrostenedione (4-OH). On the 28th day after implanting, rats were killed and the thymus, spleen, prostate gland and seminal vesicles removed for weighing and histology. In addition, estrogen receptors were measured in the thymus was enlarged after orchidectomy and greatly restored in aging rats. In aging rats, both aromatase inhibitors restored the thymus, which appeared normal histologically. In addition, ATD enlarged the thymus in young intact animals. Doses of testosterone which restored the accessory sex organs to weights measured in intact rats prevented the effects of orchidectomy on the thymus, and in old rats the effects of testosterone were blocked by ATD in both thymus and spleen. Available cytosolic estrogen receptors were reduced in thymus of testosterone-treated orchidectomized rats, and this effect blocked by ATD, which itself was apparently able to induce estrogen receptors. Receptors could not be detected in thymus from aging rats, but were measureable in cytosols from thymus of orchidectomized or ATD-treated old rats. It is therefore possible to restore the thymus in intact aging rats without recourse to surgical or chemical castration, and such a maneuver may possibly be of use to enhance an immune system weakened by aging or disease.

Ontogeny of the sexually dimorphic male nucleus in the preoptic / anterior hypothalamus of ferrets and its manipulation by gonadal steroids

A sexually dimorphic nucleus exists in the dorsal region of the ferret preoptic/anterior hypothalamic area (POA/AH), and is called the male nucleus of the POA/AH (MN-POA/AH) because it is found only in males. Development of the MN-POA/AH was studied in male ferrets, and for comparison a sexually nondimorphic ventral POA/AH nucleus was studied in both sexes. The MN-POA/AH was conspicuous in males as early as embryonic day 37 (E37) of a 41-day gestation, and its volume increased until postnatal day 56 (P56). No nucleus was present in the dorsal POA/AH of females at any age. The densities and average somal areas of cells in the dorsal POA/AH were similar in males and females at E33, before the MN-POA/AH could be visualized. However, at E37 and E41 dorsal cells were greater in density and/or somal area in males than in females, accounting for the appearance of a nucleus in males at these ages. To insure that the dorsal POA/AH nucleus seen in males at E37 and E41 was the presumptive MN-POA/AH present in adult males, pregnant ferrets were given progesterone and either implanted subcutaneously (s.c.) with testosterone (T) or ovariectomized and implanted s.c. with the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), on day 30 of gestation. As predicted from previous studies in which subjects were sacrificed in adulthood, formation of a dorsal POA/AH nucleus was promoted in female ferrets by T, and blocked in males by maternal ovariectomy and ATD treatment for animals sacrificed at E41. Much evidence suggests that behavioral sexual differentiation is accomplished in the male ferret between age E28 and P20. The MN-POA/AH is present and potentially functional in males during a considerable portion of this perinatal period.