Circulating estradiol and the activation of male and female copulatory behavior in Japanese quail (Coturnix japonica)

Previous experiments using systemic and preoptic area (POA) hormone treatments have shown that aromatization of testosterone (T) to estrogen (E) is essential for activation of male-typical copulatory behavior in castrated male Japanese quail (Coturnix japonica). Two experiments were conducted to determine whether circulating estrogen levels characteristic of normal intact males are high enough to activate male-typical or female-typical copulatory behavior. In Experiment 1, blood samples were drawn every 4 hr from groups of sexually active male quail housed under a 16L:8D light-dark cycle, and assayed for estradiol (E2) concentration. The mean +/- SEM serum E2 was 54.2 +/- 3.6 pg/ml, and no daily cycle in serum E2 was seen. The males were then tested for sexual behavior; 88% mounted females, and 23% crouched when mounted by males. In Experiment 2, 51 males were castrated and implanted with Silastic tubes containing estradiol benzoate (EB) and/or cholesterol designed to produce five different levels of serum E2, then tested for male- and female-typical copulatory behavior and bled. The serum E2 in EB-implanted quail which mounted (253 +/- 30 pg/ml) was significantly higher than that of intact quail in Experiment 1, and only 10.2% of intact males had serum E2 as high as the minimum associated with mounting in EB-implanted males. These results show that serum E2 levels in intact males are not high enough to support male-typical copulation, and that aromatization in the POA to produce locally high E2 levels may be required. In addition, it was found that the threshold serum E2 to elevate receptivity significantly was 3.6 times the intact male level, and only slightly higher than serum E2 reported for intact females. Thus the lack of receptivity in intact males is probably due to insufficient circulating E2, and the male is not defeminized with respect to sensitivity to E2 for activation of receptivity.     

Dissociation of the effects of gonadal steroids on brain serotonin metabolism and sexual behavior in the male rat

Steady-state concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in anterior hypothalamus (AH), preoptic area (POA), suprachiasmatic nucleus (SCN) and ventromedial nucleus (VMH) were equivalent 30 days after castration or sham operation in male rats killed at either the middle of the light or dark periods of a 12 : 12 h day/night cycle. Chronic administration of testosterone, 5alpha-dyhydrotestosterone (DHT), estradiol-17beta (E2) or E2+DHT for 30 days to castrated males also failed to alter differentially the concentrations of 5-HT and 5-HIAA in any of the brain regions assayed. Although castrated and sham-operated rats had similar levels of 5-HT and 5-HIAA in whole hypothalamus + preoptic area (H + POA), brain stem (BS), and cortex (CTX) when measured 7 and 32 days postoperatively, the mounting rates and the percentage of animals ejaculating were similar for both groups 6 days postoperatively whereas only the sham-operated animals continued to mount at a high rate and to ejaculate 31 days postoperatively. Also, 30 days after surgery, there were no differences among groups of males which were either castrated, castrated and given a subcutaneous capsule of testosterone, or sham operated, in the accumulation of 5-hydroxytryptophan in the H + POA, BS, or CTX measured 30 min after an intraperitoneal injection of an aromatic amino acid decarboxylase inhibitor. These results suggest the the hormonal activation of masculine sexual behavior in the rat is not mediated by a change in the metabolic activity of 5-HY neurons in either the hypothalamus or POA.     

The effect of aromatase inhibition on the sexual differentiation of the sheep brain

This study tested the hypothesis that aromatization of testosterone to estradiol is necessary for sexual differentiation of the sheep brain. Pregnant ewes (n=10) were treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) during the period of gestation when the sheep brain is maximally sensitive to the behavior-modifying effects of exogenous testosterone (embryonic d 50–80; 147 d is term). Control (n=10) ewes received vehicle injections. Fifteen control lambs (7 males and 8 females) and 17 ATD-exposed lambs (7 males and 10 females) were evaluated for sexually dimorphic behavioral and neuroendocrine traits as adults. Prenatal ATD exposure had no significant effect on serum concentrations of androgen at birth, growth rates, expression of juvenile play behaviors, or the onset of puberty in male and female lambs. Rams exposed to ATD prenatally exhibited a modest, but significant, decrease in mounting behavior at 18 mo of age. However, prenatal ATD exposure did not interfere with defeminization of adult sexual partner preferences, receptive behavior, or the LH surge mechanism. In summary, our results indicate that aromatization is necessary for complete behavioral masculinization in sheep. However, before we can conclude that aromatization does not play a role in defeminization of the sheep brain, it will be necessary to evaluate whether intrauterine exposure of male fetuses to higher doses of ATD for a more extended period of time can disrupt normal neuroendocrine and behavioral development.     

Role of androgens in the regulation of sexual behavior in the female musk shrew

These experiments were conducted to determine whether androgens are involved in the regulation of female sexual behavior in the musk shrew (Suncus murinus). In the musk shrew, sexual behavior can be reinstated after ovariectomy by the administration of supraphysiological doses of either estradiol (E2) or testosterone. However, physiological doses of E2 are not effective in this regard. To examine the role of androgens, ovariectomized (OVX) females received testosterone-filled hormone implants. These implants reinstated sexual behavior in a dose-dependent manner. To determine whether the aromatization of androgen is essential for restoration of sexual behavior, the nonaromatizable androgen, dihydrotestosterone (DHT), was administered to OVX females either alone or with an implant containing E2; sexual behavior was not restored. In the third experiment, gonadally intact females, treated with the aromatization inhibitor 1,4,6-androstatriene-3,17-dione (ATD) exhibited no sexual behavior and had significantly lower levels of aromatase activity in the medial basal hypothalamus/preoptic area than control females. In the last experiment, OVX females implanted with testosterone and given concurrent ATD treatment demonstrated significantly less sexual behavior as compared with controls. These results suggest that either estrogens produced via androgen aromatization in the brain and/or an intermediate product in the aromatization process are involved in the regulation of sexual behavior in the female musk shrew.     

Regulation of pseudosexual behavior in the parthenogenetic whiptail lizard, Cnemidophorus uniparens

Neuroendocrine mechanisms underlying complementary behaviors like male-typical mounting and female-typical receptivity are most often studied independently in males and females, respectively. Cnemidophorus uniparens is a unisexual lizard species consisting only of females that alternately express male- and female-like pseudosexual behavior across the ovarian cycle. Intact, postovulatory (PostOv), and ovariectomized (OVX), androgen-implanted animals [OVX plus testosterone (T)] exhibit male-like mounting, but not receptivity, whereas intact, preovulatory (PreOv), and OVX lizards injected with estradiol [OVX plus estrogen (E)] express receptivity, but not mounting. We tested whether the serotonergic system in the preoptic area (POA) and ventromedial nucleus of the hypothalamus (VMN) gates the reciprocal inhibition characterizing this alternating expression of mounting and receptivity. Serotonergic signaling at the POA appears to be key to gating male-like behavior. Postovulatory and OVX plus T animals have lower intracellular serotonin (5-HT) levels, and greater abundance of inhibitory 5-HT1A receptor mRNA in the POA compared with both PreOv and OVX plus E lizards. Moreover, injecting 5-HT into the POA of OVX plus T animals suppresses mounting, whereas injection into VMN of OVX plus E lizards suppresses receptivity. Although 5-HT levels in the VMN do not differ across the ovarian cycle or between hormonally manipulated animals, PreOv and OVX plus E lizards have a lower abundance of 5-HT2A mRNA in the VMN. Stimulating 5-HT1A receptors using systemic drug administration inhibits mounting, whereas activating 5-HT2A receptors facilitates receptivity. This study illuminates how male- and female-typical sexual behaviors share common neural circuits, and that 5-HT regulates these naturally complementary, and mutually exclusive, behaviors.     

Effects of estrogen in the male rat medial amygdala: infusion of an aromatase inhibitor lowers mating and bovine serum albumin-conjugated estradiol implants do not promote mating

In male rats, copulatory behavior depends on estrogen-responsive neurons located in brain areas known to be crucial for mating. Blocking the aromatization of testosterone (T) to estradiol (E(2)) either throughout the brain or within the medial preoptic area (MPO) reduces mating, whereas E(2) treatment of either the MPO or the medial amygdala (MEA) maintains sexual behavior. The effects of T aromatization in the MEA have received less attention; therefore, 2 studies were done to further elucidate the effects of E(2) in the MEA. In experiment 1, gonadally intact male rats that showed robust mating behavior were administered chronic fadrozole, a nonsteroidal aromatase inhibitor, to the MEA to stop the conversion of T to E(2) and then paired with receptive females. Infusion of fadrozole to the MEA significantly lowered mating behavior in experimental males compared to vehicle-infused control males. To further investigate the mechanism by which E(2) acts in the MEA, in experiment 2, E(2) conjugated to bovine serum albumin (BSA-E(2): a complex of E(2 )and a large protein that does not cross the plasma membrane, thereby restricting the action of E(2) to cell-surface signaling) was chronically administered bilaterally to the MEA of castrated, dihydrotestosterone-treated males. This treatment did not maintain mating behavior. These studies show that E(2) acts in the MEA to promote male sexual behavior and suggest an intercellular mechanism of E(2) action.     

Activation of sexual behaviour in castrated rats: the role of oestradiol

Sexual behaviour was induced in castrated male rats with oestradiol-17 beta- or testosterone-filled constant-release implants. Testosterone-induced sexual behaviour was unaffected by treatment with the 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one (4-MA; 16.7 mg/day) but treatment with the aromatization inhibitor 1,4,6-androstatriene-3,17-dione (ATD; 10 mg/day) prevented testosterone from inducing the behaviour. Sexual behaviour could be activated in castrated rats treated with testosterone plus ATD by treatment with 4-MA or with implants filled with a low dose of oestradiol. Lordosis behaviour induced in ovariectomized rats with testosterone-filled implants and progesterone was blocked by ATD treatment and could not be activated with 4-MA but oestradiol implants restored the display of lordosis in the testosterone plus ATD-treated females. 4-MA inhibited the in-vitro formation of [14C]5 alpha-dihydrotestosterone from [14C]testosterone by combined preoptic and hypothalamic tissue at all doses tested and a high dose of oestradiol exerted a similar effect. The results suggest that androgen aromatization is required for testosterone-activated female sexual behaviour but not for testosterone-activated male sexual behaviour. It is suggested that oestradiol normally acts to control the sexual behaviour of male rats by modifying neural androgen metabolism.     

Differentiation in male ferrets of a sexually dimorphic nucleus of the preoptic/anterior hypothalamic area requires prenatal estrogen

Experiments were conducted to determine when during perinatal development testicular steroids act in ferrets to promote the organization of a bilateral nucleus in a medial position at the border of the preoptic area (POA) and anterior hypothalamus (AH), henceforth referred to as the male nucleus of the POA/AH (MN-POA/AH). The formation of the MN-POA/AH was promoted in female offspring by treating their mothers with testosterone over the last 11 days of the 42-day gestation period, whereas MN-POA/AH formation was not disrupted in males castrated within 1, 2 or 5 days of birth. Additional experiments were conducted to determine whether the active hormone which induces differentiation of the MN-POA/AH in the male ferret is an androgen or an estrogen. MN-POA/AH formation was inhibited in males deprived prenatally of estrogenic stimulation via maternal ovariectomy and subcutaneous implantation of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) on gestational day 30. By contrast, MN-POA/AH formation was not disrupted in males exposed prenatally to the antiandrogen flutamide. These results imply that estrogen, derived from the neural aromatization of circulating testosterone, acts prenatally to promote the organization of the MN-POA/AH in male ferrets. The development of sex-dependent features of forebrain morphology may depend on the neural action of estrogen in males of diverse mammalian species.     

Implants of estradiol conjugated to bovine serum albumin in the male rat medial preoptic area promote copulatory behavior

The expression of mating behavior in male rats is dependent on estrogen-responsive neurons in the medial preoptic area (MPO). Previous reports showed that mating is attenuated if the aromatization of testosterone to estradiol (E2) is blocked in the MPO and that mating is maintained by MPO E2 implants. However, the mechanisms by which E2 exerts its action are not fully understood. It had been thought that E2 acted exclusively by binding to nuclear estrogen receptors to exert it effects; however, recent reports suggest that E2 also binds to membrane-associated receptors activating downstream intracellular cascade responses. In this study, we aimed to determine if an action of E2 at the cell surface is sufficient to support mating behavior. Therefore, either vehicle, E2, or E2 conjugated to bovine serum albumin (BSA-E2: a complex of E2 and a large protein that will not cross the plasma membrane, thereby restricting the action of E2 to cell surface signaling) was chronically administered bilaterally to the MPO of castrated, dihydrotestosterone-treated male rats. Mating behavior was supported by MPO BSA-E2 implants, suggesting that E2 operates in the MPO via a cell surface mechanism to facilitate male rat mating behavior.     

Formation of behaviorally active estrogen in the dove brain: induction of preoptic aromatase by intracranial testosterone

The preoptic area (POA) of the male ring dove is a target for specific behavioral effects of estrogen that are separable from those of androgen. Activity of the aromatase system in the POA, which converts testosterone to 17 beta-estradiol (E2), is increased by systemic androgen. Using crystalline steroid implants positioned stereotaxically in the brain, we examined whether aromatase induction is a result of direct steroid action on the POA, which can occur independently of the behavioral effects of androgen. Implants of testosterone propionate (TP) greater than 1.0 mm from POA nuclei were ineffective in increasing preoptic aromatase activity irrespective of whether the implants were unilateral, or bilateral with twice the potential output of testosterone. Within the 1.0-mm range, distance of implant from the POA nuclei was negatively correlated with induced aromatase activity in POA samples, indicating a direct effect of testosterone or its metabolites on enzyme activity. Induction of aromatase activity was higher in the right side of the POA than the left, suggesting asymmetry in inducible aromatase. Inactive 5 beta-reduced androstanes, 5 alpha-dihydrotestosterone and 17 beta-estradiol were formed from intracranial 3H-testosterone in POA. Since estradiol benzoate implants did not induce aromatase activity, this metabolite does not appear to act directly on the POA, although it is effective if administered systemically. Implants of TP in the region of the POA caused vocal behavior (perch calling) to be shown by some males. There was no correlation between the behavioral effectiveness of implants and induced POA aromatase activity. Since increase in E2 formation occurred in the absence of vocal behavior, activation of androgen-dependent behavior is not an absolute requirement for the induction effect. We conclude that testosterone can influence aromatase activity required for local production of E2 in the brain by direct action on POA cells.     

Failure of the synthetic androgen 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-triene-3-one (methyltrienolone, R1881) to duplicate the activational effect of testosterone on mating in castrated male rats

Administration of the synthetic steroid, 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-triene-3-one (methyltrienolone, R1881), which is an androgen receptor agonist not metabolized in vivo, at doses of 400, 800 or 2400 micrograms/kg s.c. in propylene glycol stimulated mounting and ejaculation only slightly in sexually experienced castrated rats. A similar low level of mating was observed in a group of castrated rats given testosterone at doses of 400 followed by 800 micrograms/kg. However, when treated with a higher dose of testosterone (2400 micrograms/kg) these castrated rats displayed significantly higher levels of mounting and ejaculation than rats treated with any of the doses of R1881. Also, when this higher dosage of testosterone was substituted for each dose of R1881, significant increments in mounting and ejaculation occurred in all groups. These findings show that R1881 is only marginally effective in restoring sexual behaviour in castrated rats, suggesting that the activation of neural androgen receptors cannot by itself account for the activational effect of testosterone on mating behaviour in gonadally intact male rats.     

Combined aromatase inhibitor and antiandrogen treatment decreases territorial aggression in a wild songbird during the nonbreeding season.

Male song sparrows (Melospiza melodia morphna) defend territories throughout the year in western Washington State. In the nonbreeding season (autumn and winter), aggression and song are robustly expressed but plasma testosterone (T) levels are basal. Also, castration does not decrease nonbreeding territoriality. In this field experiment, we asked whether nonbreeding aggression is independent of T. T can act via androgen receptors or T can be aromatized to 17beta-estradiol (E(2)) and act via estrogen receptors. We treated free-living nonbreeding birds with an aromatase inhibitor (ATD) and an androgen receptor antagonist (flutamide) in combination. We then challenged subjects with a live decoy and playback of tape-recorded songs. ATD+flutamide treatment decreased several aggressive behaviors. However, ATD+flutamide treatment did not affect body condition, suggesting that subjects were healthy and that foraging behavior was not reduced. As expected, ATD+flutamide treatment increased plasma T, probably by blocking negative feedback on luteinizing hormone. Surprisingly, ATD+flutamide treatment increased plasma E(2). Most other studies using aromatase inhibitors have not measured plasma E(2). However, it is possible that ATD+flutamide treatment decreased local E(2) levels in the brain but not in plasma. Finally, ATD+flutamide treatment increased plasma corticosterone, perhaps in response to increased plasma T or E(2). To our knowledge, these are the first data to suggest that nonbreeding territoriality is regulated by endogenous steroid hormones. Nongonadal production of sex steroids may support aggression in the nonbreeding season.     

Pro-opiomelanocortin messenger RNA in hypothalamic neurons is increased by testosterone through aromatization to estradiol

We have previously demonstrated that neurons in the rostral arcuate nucleus expressing the messenger RNA (mRNA) for pro-opiomelanocortin (POMC) are responsive to modulation by physiological levels of testosterone. It is uncertain, however, whether testosterone's action is mediated through direct activation of androgen receptors or through aromatization to estradiol and subsequent binding to estrogen receptors. We examined this question by evaluating the effectiveness of estradiol and dihydrotestosterone (DHT), a nonaromatizable androgen, in reversing the castration-induced diminution of POMC mRNA in the arcuate nucleus. Using in situ hybridization, we measured POMC mRNA content within arcuate neurons of intact, castrated, castrated testosterone-replaced, castrated estradiol-replaced, and castrated DHT-replaced male rats. Adult male rats were castrated and implanted (s.c.) with a Silastic capsule filled to one of the following specifications: crystalline testosterone (30 mm; n = 4); 17 beta-estradiol (E2) diluted 1:1 with cholesterol (5 mm; n = 4); DHT (40 mm; n = 4); or empty (30 mm; n = 4). Control, sham-operated animals (n = 4) were left intact. Analysis of the results showed that following castration, POMC mRNA content was significantly reduced in cells of the arcuate nucleus (intact: 152 +/- 3 grains/cell vs. castrate: 110 +/- 3 grains/cell). Replacement with physiological levels of testosterone prevented the decline of POMC mRNA levels (castrated testosterone-replaced: 143 +/- 6 grains/cell), as did replacement with physiological levels of estrogen (castrated estrogen-replaced: 149 +/- 8 grains/cell). Treatment with DHT failed to prevent the postcastration decline in POMC mRNA content (castrated DHT-treated: 118 +/- 4 grains/cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of androgens on hypothalamic pro-opiomelanocortin

Testosterone and estradiol have been shown to affect the hypothalamic content of several pro-opiomelanocortin (POMC)-derived peptides in castrated male and female rats, respectively. It was unclear, however, whether the effects of testosterone on hypothalamic POMC were due to conversion by aromatization to estradiol or whether there were independent androgen actions on hypothalamic POMC. In order to answer this question, the effect of treatment with the nonaromitizable androgen 5-alpha-dihydrotestosterone (DHT) on the concentration of beta-endorphin (beta-EP) in the medial basal hypothalamus (MBH) was studied in castrated male rats and compared to the effect of treatment with testosterone or estradiol. The concentrations of two other POMC-derived peptides, corticotropin-like intermediate lobe peptide (CLIP) and alpha-MSH were measured as well. Adult male rats were castrated and received either no treatment or treatment with subcutaneously implanted silastic capsules, containing either DHT, testosterone or estradiol, designed to produce steroid levels in a physiological range. After 4 weeks the mean concentration of beta-EP in the MBH of the untreated castrated rats was 1,640 +/- 56 fmol/mg protein. This was reduced significantly to 1,184 +/- 74 fmol/mg protein after DHT treatment (p less than 0.001). Similar reductions to 1,340 +/- 95 and 1,130 +/- 85 fmol/mg protein were noted after testosterone and estradiol treatment, respectively. The mean CLIP concentration of 1,870 +/- 73 fmol/mg protein in the untreated animals fell to 1,390 +/- 95 after DHT (p less than 0.001) compared to 1,520 +/- 105 and 1,260 +/- 101 after testosterone and estradiol treatment, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Gonadotrophin and prolactin secretion in castrated male sheep following subcutaneous or intracranial treatment with testicular hormones

Interactions between testosterone, estradiol, and inhibin in the control of gonadotrophin secretion in males are poorly understood. Castrated rams were treated with steroid-free bovine follicular fluid (bFF), testosterone, or estradiol and for 7 d(2×2×2 factorial design). Given independently, none of the exogenous hormones affected follicle-stimulating hormone (FSH) concentrations, but the combination of one or both steroids with bFF reduced FSH secretion. Testosterone and estradiol reduced luteinizing hormone (LH) pulse frequency (there was no synergism), and bFF had no effect. Plasma prolactin concentrations were not affected by any treatment. To locate the central sites of steroid action, castrated rams were bilaterally implanted in the preoptic area (POA), ventromedial nucleus (VMH), or arcuate nucleus (ARC). These implants did not affect FSH or prolactin concentrations, or LH pulse amplitude. The frequency of the LH pulses was not affected by testosterone in any site. Estradiol located in the ARC, but not the POA or VMH, decreased LH pulse frequency. In summary, FSH secretion is controlled by synergistic interactions between inhibin and estradiol or testosterone, whereas GnRH/LH pulse frequency is controlled by testicular steroids. Estradiol acts partly, at least, in the ARC, but the central site of action, testosterone remains unknown.     

Evidence for a role of testosterone-androgen receptor interactions in mediating masculine sexual behavior in male rats

The purpose of this study was 2-fold: 1) to use gonadal steroid hormone exposures in the physiological range to assess the relative roles of testosterone (T), estradiol (E2), and dihydrotestosterone (DHT) in the expression of male sexual behavior, and 2) to determine whether androgen receptor (AR) or estrogen receptor (E2R) occupation is increased after exposure to these various gonadal steroid hormones. Sexually experienced, castrated male rats implanted sc with Silastic capsules containing T, 10% E2, DHT, 10% E2 plus DHT, or blanks provided hormone levels in the physiological range. Copulatory behavior was measured on days 2-4, 5-7, 10-12, and 14-16 of steroid treatment. Although T, E2, and E2 plus DHT treatments all activated mounting, only T was effective in restoring ejaculation in 100% of the males. DHT alone had no effect on any aspect of male sexual behavior. Brains of males given these various hormone treatments were assayed for both cell nuclear AR and cell nuclear E2R binding in the hypothalamus, preoptic area, amygdala, and septum. Results indicate that when hormone levels in the physiological range were employed, T and DHT bind primarily to AR, whereas E2 binds to E2R. In a second experiment, 0.5% E2 plus DHT was found to yield AR and E2R levels comparable to those in rats receiving T capsules. Male rats bearing these capsules showed virtually no sexual behavior, demonstrating that elevation of AR and E2R levels comparable to those generated by T is not sufficient to induce male sexual behavior. We then measured intact AR and E2R levels and determined that in intact males E2R levels were higher than in T-treated males. These E2R levels could be replicated using 1.0% E2. Males exposed to 1.0% E2 plus DHT failed to display male sexual behavior. These data suggest that 1) relatively high and prolonged levels of E2R occupation are required for estrogen activation of male sexual behavior, 2) high levels of AR occupation induced by DHT are not sufficient to activate male sexual behavior, and 3) in intact male rats T, acting via androgen receptors, plays a primary role in mediating the expression of masculine sexual behavior.     

Formation of behaviorally effective 17 beta-estradiol in the dove brain: steroid control of preoptic aromatase

The preoptic area (POA) of the male dove is a known target area for separable behavioral actions of testosterone and 17 beta-estradiol (E2) and contains an active aromatase system. We have examined the regulatory influence of gonadal hormones on aromatase using an in vitro microassay that measures conversion of [1 alpha, 2 alpha-3H]testosterone to E2 in anatomically defined brain samples of individual animals. Preoptic aromatase activity, which is higher in males than females, is decreased (77%) in both short (30 day) and long term (180 day) castrates, indicating that gonadal hormones maintain POA aromatase activity. Basal levels of POA activity are not influenced by the period of hormonal deficit. Low levels of aromatase activity detected in area basalis are also unaffected by castration. Intramuscular testosterone propionate rapidly increases aromatase activity (within 12 h) specifically in POA of castrated males. The inductive effect of testosterone propionate in castrated doves is not increased by limited behavioral interactions in a test situation with sexually active females. A nonaromatizable androgen, 5 alpha-dihydrotestosterone, has no effect on POA aromatase activity, whereas the activity of this enzyme is restored to levels of sexually active males by systemic E2. Diethylstilbestrol has a similar, though less potent effect, indicating an estrogenic action on the enzyme. We conclude that circulating androgen modulates preoptic aromatase activity. The product of the reaction, E2, is also likely to be involved as part of a positive feedback system.     

Brain aromatization of testosterone in the male Syrian hamster: effects of androgen and photoperiod

Estrogen formed by aromatization of testosterone (T) is involved in the activation of sexual behavior and control of the neuroendocrine system in the male Syrian hamster. Our study examined whether daylength influences formation of estrogen in the preoptic area (POA) and other neuroendocrine areas (anterior hypothalamus, AHT, and medial amygdala, MA) which are targets for T in behaviorally active males. Estrogen formation in individual brain samples was assayed in vitro using the stereospecific production of 3H2O from (1 beta-3H) T as a measurement of aromatase activity. Serum levels of PRL, LH, FSH and T were compared with brain aromatase activity. Groups of intact, castrated and T-treated (chronic silastic T implants) male hamsters, previously selected on behavioral criteria as being sexually active, were maintained on long (16:8LD) or short (8:16LD) daylength for 16 weeks. Two further groups of males either intact or castrated and T-treated were shifted after 7 weeks from the long photoperiod to 12:12LD. POA, AHT and MA areas of sexually active males contained active aromatase systems which converted 3H-T to estrogens. Enzyme activity differed between the areas (POA, MA greater than AHT). Aromatase activity was low in medial septum and cerebral samples. Castration, which reduced serum T to undetectable levels and elevated LH and FSH, had no effect on preoptic aromatase activity. Although estrogen formation in POA did not differ between 8:16LD and 16:8LD males, castration reduced aromatase activity in AHT of both short- and long-day groups. Aromatase activity in AHT was also significantly reduced in photo-inhibited 12:12LD intact males. There was no analogous decrease in 5 alpha-reductase or 17 beta-hydroxysteroid dehydrogenase (HSD) activity, indicating a specific effect on the aromatase. The effect of photoperiod on aromatase activity was not reversed by T treatment. Therefore, photoinhibition acts in part through the effects of reduced T levels on the anterior hypothalamus, but it also acts independently of circulating T. Our results suggest that both androgen and photoperiod may regulate the AHT aromatase system and that this occurs by different mechanisms. The more active aromatase system in POA is insensitive to both castration and photoperiod. Behavioral deficits in short-day males are not due to changes in the preoptic aromatase system, but may be related to changes in aromatase activity within AHT. We conclude that there is a difference in the regulation of two locally active aromatase systems within the preoptic-anterior hypothalamic complex of the male hamster.     

Regulation of luteinizing hormone secretion in male sheep by endogenous estrogen

Both androgens and estrogens are implicated in the regulation of gonadotropin secretion in the male. Three experiments were conducted to determine the physiological importance of estradiol (E2) in the feedback regulation of LH secretion in male sheep. In the first experiment, LH secretion in castrate rams (wethers) was shown to be susceptible to the picomolar concentrations of E2 found in intact rams. In the second experiment, aminoglutethimide (AG) was administered to testosterone-implanted wethers to block aromatization of testosterone and ascertain the possible consequences of E2 deprivation on testosterone-mediated LH suppression. AG had no apparent effect on serum testosterone, but reduced serum E2 by half and increased serum LH approximately 3-fold. These data suggest that aromatization of testosterone to E2 is a physiologically important step in the regulation of LH secretion in the ram and that aromatization occurs, at least in part, in peripheral tissues. The relative contributions of peripherally and centrally derived E2 in the regulation of LH remain uncertain. In the third experiment, immunoneutralization of endogenous E2 in intact rams by active immunization against E2 was shown to stimulate pulsatile LH secretion and elevate basal and mean LH concentrations. Associated with this LH response was a significant increase in serum testosterone, such that immunized rams exposed to a nonstimulatory 16-h light, 8-h dark photoperiod had testosterone concentrations equivalent to those of control rams exposed to a stimulatory 8-h light, 16-h dark photoperiod. Together, these findings emphasize the importance of E2 in the control of male reproduction and suggest the possibility of improving year-round reproductive performance of the domestic ram through E2 immunoneutralization.