Fos Expression in the Olfactory Pathway of High- and Low-Sexually Performing Rams Exposed to Urine from Estrous or Ovariectomized Ewes.

Exposure to estrous ewe urine stimulates investigation and mounting activity in sexually active but not sexually inactive rams. It was hypothesized sexual indifference may result from an inability to detect olfactory cues or an interruption of the pathway from detection of the olfactory stimulus to the motor response. Sexually active (n=4) and inactive (n=3) rams were exposed to urine from estrous ewes. An additional group of sexually active rams (n=3) were exposed to urine from ovariectomized ewes. Rams were exsanguinated following 1 h of exposure to stimulus. Neural activity was determined in tissues of interest by the presence of fos and fos-related proteins detected by immunohistochemistry procedures. Sexually active rams exposed to urine from ovariectomized ewes had more (P ≤ 0.05) fos-positive cells in the olfactory bulb, but fewer (P = 0.03) fos-positive cells in the cortical amygdala compared to sexually active rams exposed to urine from estrous ewes. Sexually inactive rams had similar (P ≥ 0.13) numbers of fos positive neurons in the olfactory bulb and medial amygdala but fewer (P ≤ 0.04) in the central amygdala, bed nucleus of the stria terminalis and the medial preoptic area compared to sexually active rams exposed to urine from estrous ewes. Sexual inactivity was not associated with decreased hypothalamic function since fos activity was similar (P ≥ 0.14) among groups in the suprachiasmatic and ventral medial nucleus. Sexual inactivity is not likely due to an impaired ability to detect or process olfactory stimuli by the main olfactory bulb and medial-cortical amygdala. Sexually inactive rams may have reduced attentiveness to sexual stimuli and/or decreased responsiveness of regions in the brain which regulate reproductive behaviors.

Porcine hypothalamic aromatase cytochrome P450: isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars.

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Changes in LH secretion in response to an estradiol challenge in male- and female-oriented rams and in ewes.

Two experiments were conducted to determine whether an estradiol challenge could cause a female-type LH surge in castrated male- and female-oriented rams (MORs and FORs). Administration of 17beta-estradiol to castrated MORs and FORs and ovariectomized ewes caused an initial reduction in LH secretion followed for 12-20 h by a surge release of LH in the ewes. No surge release of LH occurred in the MORs and FORs. The pattern of changes in LH secretion within rams and ewes did not differ between the breeding and nonbreeding seasons. Treatment failed to elicit female-typical receptive sexual behaviors in the rams but did stimulate increased sexual receptivity in the ewes as determined by the measures of responsiveness to the teaser ram. Overall, no differences were found in hypothalamic-hypophyseal function in response to exogenous estradiol between MORs and FORs. These data are interpreted to suggest that in contrast to sexual attraction, the neural mechanisms controlling the LH surge and female receptivity are defeminized in MORs.

Neurobiology of gender identity and sexual orientation.

Sexual identity and sexual orientation are independent components of a person's sexual identity. These dimensions are most often in harmony with each other and with an individual's genital sex, although not always. The present review discusses the relationship of sexual identity and sexual orientation to prenatal factors that act to shape the development of the brain and the expression of sexual behaviours in animals and humans. One major influence discussed relates to organisational effects that the early hormone environment exerts on both gender identity and sexual orientation. Evidence that gender identity and sexual orientation are masculinised by prenatal exposure to testosterone and feminised in it absence is drawn from basic research in animals, correlations of biometric indices of androgen exposure and studies of clinical conditions associated with disorders in sexual development. There are, however, important exceptions to this theory that have yet to be resolved. Family and twin studies indicate that genes play a role, although no specific candidate genes have been identified. Evidence that relates to the number of older brothers implicates maternal immune responses as a contributing factor for male sexual orientation. It remains speculative how these influences might relate to each other and interact with postnatal socialisation. Nonetheless, despite the many challenges to research in this area, existing empirical evidence makes it clear that there is a significant biological contribution to the development of an individual's sexual identity and sexual orientation.

Neuropeptide and steroid hormone mediators of neuroendocrine regulation.

To maintain the health and well-being of all mammals, numerous aspects of physiology are controlled by neuroendocrine mechanisms. These mechanisms ultimately enable communication between neurones and glands throughout the body and are centrally mediated by neuropeptides and/or steroid hormones. A recent session at the International Workshop in Neuroendocrinology highlighted the essential roles of some of these neuropeptide and steroid hormone mediators in the neuroendocrine regulation of stress-, reproduction- and behaviour-related processes. Accordingly, the present review highlights topics presented in this session, including the role of the neuropeptides corticotrophin-releasing factor and gonadotrophin-releasing hormone in stress and reproductive physiology, respectively. Additionally, it details an important role for gonadal sex steroids in the development of behavioural sex preference.

Early prenatal androgen exposure reduces testes size and sperm concentration in sheep without altering neuroendocrine differentiation and masculine sexual behavior.

Prenatal androgens are largely responsible for growth and differentiation of the genital tract and testis and for organization of the control mechanisms regulating male reproductive physiology and behavior. The aim of the present study was to evaluate the impact of inappropriate exposure to excess testosterone (T) during the first trimester of fetal development on the reproductive function, sexual behavior, and fertility potential of rams. We found that biweekly maternal T propionate (100 mg) treatment administered from Day 30-58 of gestation significantly decreased (P < 0.05) postpubertal scrotal circumference and sperm concentration. Prenatal T exposure did not alter ejaculate volume, sperm motility and morphology or testis morphology. There was, however, a trend for more T-exposed rams than controls to be classified as unsatisfactory potential breeders during breeding soundness examinations. Postnatal serum T concentrations were not affected by prenatal T exposure, nor was the expression of key testicular genes essential for spermatogenesis and steroidogenesis. Basal serum LH did not differ between treatment groups, nor did pituitary responsiveness to GnRH. T-exposed rams, like control males, exhibited vigorous libido and were sexually attracted to estrous females. In summary, these results suggest that exposure to exogenous T during the first trimester of gestation can negatively impact spermatogenesis and compromise the reproductive fitness of rams.

Neuronal activity of aromatase enzyme in non-copulating male rats.

There are apparently normal male rats that fail to initiate copulation; these animals are called non-copulating (NC) males. Several research groups have demonstrated that conversion of testosterone to oestradiol (aromatisation) in specific brain areas known to be involved in the control of masculine sexual behaviour is fundamental in the control of masculine sexual behaviour. The aim of the present study was to test the hypothesis that the concentration of aromatase activity (AA) in the brain is lower in NC males than in copulating males (C). We quantified AA in several brain nuclei and also evaluated whether NC rats have altered concentrations of testosterone in their plasma. We found that AA was reduced in the medial preoptic nuclei (MPN) of NC male rats vs C males. In addition, NC and C male rats had similar plasma levels of testosterone. These data suggest that reduced levels of AA in the MPN could be a crucial factor associated with lack of male coital behaviour in rats.

Tyrosine hydroxylase in the ventral tegmental area of rams with high or low libido-A role for dopamine.

Dopamine synthesis in the ventral tegmental area (VTA) is necessary for the reinforcement of sexual behavior. The objective of this study determined if sexual stimuli initiates reward, and whether reward is attenuated in sexually inactive rams. Sexually active rams were exposed to urine from estrous (n=4) or ovariectomized (n=3) ewes with inactive rams (n=3) exposed to urine from estrous ewes. Following exposure, rams were exsanguinated and brains perfused. Alternating sections of the VTA were stained for Fos related antigens (FRA), tyrosine hydroxylase, and dopamine beta-hydroxylase activity. Forebrain tissue, mid-sagittal ventral to the anterior corpus callosum, was stained for dopamine D2 receptors. Concentrations of cortisol was determined prior to and following exposure. Exposure to ovariectomized-ewe urine in sexually active rams did not influence (P=0.6) FRA expression, but fewer (P<0.05) neurons were positive for tyrosine hydroxylase in the VTA. Sexually inactive rams had fewer (P<0.05) FRA and tyrosine hydroxylase positive neurons in the VTA than sexually active rams following exposure to estrous ewe urine. VTA neurons staining positive for dopamine beta-hydroxylase did not differ by sexual activity (P=0.44) or urine exposure (P=0.07). Exposure to stimulus did not influence (P=0.46) numbers of forebrain neurons staining positive for dopamine D2 receptors in sexually active rams, but fewer (P=0.04) neurons stain positive in inactive rams. Serum concentrations of cortisol did not differ (P≥0.52) among rams prior to or following stimulus. In conclusion sexual inactivity is unlikely due to stress, but may be partially a result of decreased tyrosine hydroxylase and/or the response to dopamine.

Effects of Long-Term Flutamide Treatment During Development on Sexual Behaviour and Hormone Responsiveness in Rams.

Testosterone exposure during midgestation differentiates neural circuits controlling sex-specific behaviours and patterns of gonadotrophin secretion in male sheep. Testosterone acts through androgen receptors (AR) and/or after aromatisation to oestradiol and binding to oestrogen receptors. The present study assessed the role of AR activation in male sexual differentiation. We compared rams that were exposed to the AR antagonist flutamide (Flu) throughout the critical period (i.e. days 30-90 of gestation) to control rams and ewes that received no prenatal treatments. The external genitalia of all Flu rams were phenotypically female. Testes were positioned s.c. in the inguinal region of the abdomen, exhibited seasonally impaired androgen secretion and were azospermic. Flu rams displayed male-typical precopulatory and mounting behaviours but could not intromit or ejaculate because they lacked a penis. Flu rams exhibited greater mounting behaviour than control rams and, similar to controls, showed sexual partner preferences for oestrous ewes. Neither control, nor Flu rams responded to oestradiol treatments with displays of female-typical receptive behaviour or LH surge responses, whereas all control ewes responded as expected. The ovine sexually dimorphic nucleus in Flu rams was intermediate in volume between control rams and ewes and significantly different from both. These results indicate that prenatal anti-androgen exposure is not able to block male sexual differentiation in sheep and suggest that compensatory mechanisms intervene to maintain sufficient androgen stimulation during development.

Developmental and Functional Effects of Steroid Hormones on the Neuroendocrine Axis and Spinal Cord.

This review highlights the principal effects of steroid hormones at central and peripheral levels in the neuroendocrine axis. The data discussed highlight the principal role of oestrogens and testosterone in hormonal programming in relation to sexual orientation, reproductive and metabolic programming, and the neuroendocrine mechanism involved in the development of polycystic ovary syndrome phenotype. Moreover, consistent with the wide range of processes in which steroid hormones take part, we discuss the protective effects of progesterone on neurodegenerative disease and the signalling mechanism involved in the genesis of oestrogen-induced pituitary prolactinomas.

Sex differences in androgen receptors and aromatase activity in microdissected regions of the rat brain.

Males are generally more responsive than females to the behavioral and neuroendocrine actions of androgens. The present experiments were performed to determine whether these differences may result from sex differences in the number of androgen receptors (AR) in specific brain areas. For this reason, AR binding was compared in both cytosol (ARc) and cell nuclear KCl extracts (ARn) from microdissected brain regions of gonadectomized male and female rats treated with doses of testosterone (T) that produced equivalent physiological circulating androgen levels. In addition, microsomal aromatase activity was measured as a biochemical index of tissue responsiveness to T, since estrogen formation in certain brain areas is regulated by androgen. One week after exogenous T administration, males exhibited significantly higher levels of ARn than females in the bed nucleus of the stria terminalis, periventricular preoptic area, and ventromedial nucleus. Males also had significantly higher aromatase levels in these same areas plus the medial preoptic nucleus and anterior hypothalamus. There were no significant differences in ARn concentrations in eight other nuclei that were examined for significant sex differences in ARc levels observed under these experimental conditions. When ARc levels were compared in untreated gonadectomized male and female rats, males had greater levels of ARc in the bed nucleus of the stria terminalis only, indicating that new receptor synthesis may be responsible for the sex differences observed in T-treated rats. These results suggest that sex differences in neural responsiveness to androgens may be due in part to sex differences in ARn occupation in specific brain regions.

Sexual differentiation of aromatase activity in the rat brain: effects of perinatal steroid exposure.

Androgens regulate aromatase activity in the medial preoptic area and other components of the brain circuit that mediates male sexual behavior. The levels of aromatase activity within these brain regions are greater in males than in females. As the activation of copulation requires aromatization of testosterone to estradiol, this quantitative enzymatic difference between sexes could contribute to the greater behavioral response displayed by males. The present study was designed to test the hypothesis that gender differences in brain aromatase activity of adult rats are dependent on the sexual differentiation of the brain that occurs during perinatal exposure to gonadal hormones. Aromatase activity was measured in vitro in microdissected brain samples using a sensitive radiometric assay. We examined the effect of pre- and postnatal treatment with testosterone propionate or diethylstilbestrol on basal levels and androgen responsiveness of aromatase in adults. In addition, we examined what effect prepubertal gonadectomy exerts on enzyme regulation. Our results demonstrate that perinatal treatments with gonadal hormones that are known to differentiate sexual behavior can completely masculinize the capacity for aromatization in the adult female. The process that differentiates aromatase expression appears to depend on androgen exposure and, in part, local estrogen synthesis, as diethylstilbestrol was able to substitute for testosterone propionate. We also observed that prepubertal gonadectomy reduced the levels of aromatase activity measured in adult brain, suggesting that gonadal hormones that are secreted during puberty may enhance the expression of aromatase activity in adulthood. From this study, we conclude that testosterone and/or its estrogenic metabolites act on the developing brain to determine the gender-specific capacity for aromatization and to regulate androgen responsiveness within components of the neural circuitry that mediates male sexual behavior.

Androgens regulate brain aromatase activity in adult male rats through a receptor mechanism.

We studied the regulation of aromatase activity in the hypothalamus-preoptic area ( HPOA ) of adult male rats using a sensitive in vitro assay which measures the amount of 3H2O formed by tissue homogenates during the conversion of [1 beta-3H]androstenedione to estrone. After castration, HPOA aromatase activity was decreased by 60% (P less than 0.05), seminal vesicle (SV) and ventral prostate (VP) weights were significantly decreased (P less than 0.05), and serum LH levels were elevated. We found that testosterone (T) or 5 alpha-dihydrotestosterone (DHT) administered in Silastic capsules for 7 days reversed the effects of castration. Testosterone and DHT stimulated HPOA aromatase activity 133% and 92%, respectively (P less than 0.05). Both steroids significantly increased SV and VP wet weights and suppressed serum levels of LH (P less than 0.05). Administration of either estradiol or progesterone did not reverse the effect of castration on HPOA aromatase activity or any other parameter measured. To determine the involvement of androgen receptors in the mechanism by which androgens affect brain aromatase, we administered the nonsteroidal antiandrogen flutamide to intact male rats (15 mg/day for 7 days). There was 42% less HPOA aromatase activity in treated rats than in oil-injected controls (P less than 0.05). Flutamide significantly decreased SV and VP wet weights, while serum LH levels were enhanced (P less than 0.05). Likewise, administration of flutamide to T-implanted castrated males blocked the T-induced increase in HPOA aromatase activity and accessory sexual organ wet weights, and prevented the T-induced suppression of serum LH. Flutamide given alone to castrated rats had no effect. Since both T and DHT stimulated HPOA aromatase activity and since the effects of T are blocked by the concomitant administration of the antiandrogen flutamide, we concluded that the control of HPOA aromatase activity by androgens is receptor mediated.

Genetic evidence for androgen-dependent and independent control of aromatase activity in the rat brain.

To investigate the role of androgen receptors in the regulation of brain aromatase activity (AA) in adult rats, the levels of AA in discrete brain areas of androgen-insensitive testicular feminized (Tfm) rats were compared with those in their normal male littermates (NL). AA was measured in homogenates of brain tissue by using a radiometric assay that quantifies the production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. Initially, we assessed the capability of block-dissected tissues to aromatize androgens. We found that the AA in the amygdala and hypothalamus-preoptic area of Tfm rats was significantly lower (P less than 0.001) than the AA in NL despite the fact that circulating androgen concentrations in the Tfm were significantly higher. Kinetics studies demonstrated that the apparent Michaelis constant was equivalent for both groups (0.02-0.03 microM). Administration of testosterone propionate to castrated males produced 3 to 4-fold elevations of AA in NL, but did not affect brain AA in Tfm rats. To pinpoint specific sites where AA is affected in Tfm rats, we measured AA in 10 hypothalamic and limbic nuclei that were dissected from 300-micron frozen brain sections. Compared to NL, Tfm rats exhibited significantly lower levels of AA in all micro-dissected brain regions studied, except for the medial and cortical amygdala. These data provide genetic evidence for both androgen-dependent and independent regulation of AA in the rat brain.

Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system.

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.

Testosterone regulates progonadotropin-releasing hormone levels in the preoptic area and basal hypothalamus of the male rat.

GnRH is synthesized as part of a larger 92-amino acid prohormone (pro-GnRH). In the present study, we examined the effect of gonadectomy and hormone replacement on the processing of pro-GnRH to GnRH in adult male rats. Immunoreactive levels of pro-GnRH and GnRH in acid extracts of preoptic area (POA) and basal hypothalamus (BH) from intact, castrate, and testosterone (T)-treated castrate males were quantified by RIA. In addition, we used immunocytochemistry to detect pro-GnRH-and GnRH-positive neurons and determine the effects of hormone treatment. Three weeks after castration, the pro-GnRH content of the POA was 2-fold greater than the amount found in intact males. Treatment with T for 1 week lowered the prohormone content to intact levels. Although the pro-GnRH content in the BH was about 50% lower than that in the POA, the BH responded to castration and steroid replacement in a manner identical to the POA. The GnRH content of the BH, but not that of the POA, was significantly reduced after castration and increased by T treatment. On the other hand, the total number of pro-GnRH and GnRH cell bodies visualized by immunocytochemistry was not significantly altered by either treatment. These results show that changes in pro-GnRH content vary in inverse relation to changes in GnRH content and suggest that gonadectomy inhibits the enzymatic processing of precursor, while T treatment promotes it.

Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male rhesus macaques.

The conversion of androgens to active metabolites by neural tissue is believed to be an essential component in the cellular mechanism of androgen-induced neuroendocrine responses. In this study, we measured the in vitro aromatization and 5 alpha-reduction of androgens in incubations of microdissected brain regions from four intact and five castrated (6 weeks) adult male rhesus monkeys. Individual nuclei were microdissected from 600-microns frozen brain sections and homogenized in a potassium phosphate buffer. Aromatase activity was measured by a radiometric assay that uses the incorporation of tritium from [1 beta-3H]androstenedione into 3H2O as an index of estrogen formation. We estimated 5 alpha-reductase activity by isolating 5 alpha-dihydrotestosterone on two different chromatography systems and measuring the amount of this product formed from [1 alpha,2 alpha-3H]testosterone. We acidified a portion of each homogenate and determined LHRH content by RIA. Between brain nuclei, aromatase activity varied 1500-fold, whereas 5 alpha-reductase activity varied only 3-fold. Both enzyme activities were highest in amygdaloid, medial preoptic, and medial diencephalic nuclei and lowest in the caudate nucleus. Aromatase activities in the supraoptic nucleus, periventricular area, medial preoptic area-anterior hypothalamus, and lateral hypothalamus were significantly (P less than 0.05) lower in castrated males. Castration did not significantly affect 5 alpha-reductase activity, except for an increase in the basolateral amygdala. The highest concentrations of LHRH were in the infundibular nucleus-median eminence and were 30 times greater than amounts measured in preoptic and medial hypothalamic nuclei. The LHRH contents of the infundibular nucleus-median eminence, ventral medial nucleus, and lateral hypothalamus were significantly lower in castrated males (P less than 0.05). In addition, we observed a significant correlation between aromatase activity and LHRH content in the basal hypothalamus of intact males (r = 0.947; P less than 0.05; n = 8), but not in the preoptic-anterior hypothalamus (r = 0.068; P greater than 0.05; n = 10). No correlation was observed between 5 alpha-reductase activity and LHRH content in either area. These data indicate that castration selectively affects androgen metabolism and LHRH content in discrete regions of the brain of male monkeys and suggest that aromatase and 5 alpha-reductase are regulated differentially in the primate brain.

The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment.

Estrogen and androgen synergize in the regulation of various neuroendocrine functions. To determine a potential cellular basis of this synergism, we measured androgen receptor (AR) in the cytosol of 16 hypothalamic and limbic nuclei and subregions in castrated male rats and castrated rats treated with estradiol. Androgen receptor was measured by a previously validated in vitro binding assay using the synthetic androgen methyltrienolone [( 3H]R1881). Male Sprague-Dawley rats (250-350 g) were castrated 2 weeks before the implantation of a 2.5-cm Silastic capsule filled with crystalline 17 beta-estradiol. Control rats were sham implanted. Estrogen treatment lasted for 1 week, after which time the animals were killed, their brains were frozen and sectioned, and individual nuclei and subregions were removed by a tissue punch technique. Tissue from six rats were combined for each determination. The highest levels of AR were found in the ventromedial nucleus (16.5 +/- 1.4 fmol/mg protein), medial preoptic area (12.1 +/- 1.4 fmol/mg protein), bed nucleus of the stria terminalis (11.6 +/- 1.4 fmol/mg protein), lateral septum (11.4 +/- 1.4 fmol/mg protein), arcuate nucleus-median eminence (10.9 +/- 2.1 fmol/mg protein), and medial amygdala (10.3 +/- 0.9 fmol/mg protein). Estrogen treatment resulted in significant increases in AR in medial preoptic area (14.8 +/- 0.6 fmol/mg protein; P less than 0.05) and medial amygdala (14.6 +/- 1.2 fmol/mg protein; P less than 0.02). Subsequent studies using block-dissected hypothalamus-preoptic area, anterior pituitary, and prostate revealed significant estrogen-mediated elevations in AR in anterior pituitary cytosol [42.2 +/- 3.0 vs. 26.4 +/- 1.6 fmol/mg protein (control); P less than 0.01], but not in hypothalamus-preoptic area or prostate cytosols. Estrogen treatment had no effect on AR affinity. The binding of [3H]R1881 was specific for AR and was not affected by the addition of radioinert progesterone to the incubation tube. Estimates of AR concentration were similar regardless of whether [3H]R1881 or [3H]dihydrotestosterone was used as the ligand. In this study, we describe the distribution of AR throughout the hypothalamus and limbic areas using biochemical techniques. In addition, we have identified some cellular events that may mediate the synergistic actions of estrogen and androgen on the neuroendocrine system.

Regulation of brain aromatase activity in rats.

We studied the distribution and regulation of aromatase activity in the adult rat brain with a sensitive in vitro assay that measures the amount of 3H2O formed during the conversion of [1 beta-3H]androstenedione to estrone. The rate of aromatase activity in the hypothalamus-preoptic area (HPOA) was linear with time up to 1 h, and with tissue concentrations up to 5 mgeq/200 microliters incubation mixture. The enzyme demonstrated a pH optimum of 7.4 and an apparent Michaelis-Menten constant (Km) of 0.04 microns. We found the greatest amount of aromatase activity in amygdala and HPOA from intact male rats. The hippocampus, midbrain tegmentum, cerebral cortex, cerebellum, and anterior pituitary all contained negligible enzymatic activity. Castration produced a significant decrease in aromatase activity in the HPOA (P less than 0.001), but not in the amygdala or cerebral cortex (P greater than 0.05). The HPOAs of male rats contained significantly greater aromatase activity than the HPOAs of female rats. In females, this enzyme activity did not change during the estrous cycle or after ovariectomy. Administration of testosterone to gonadectomized male and female rats significantly enhanced HPOA aromatase activities (P less than 0.05) to levels approximating those found in HPOA from intact males. Therefore, our results suggest that testosterone, or one of its metabolites, is a major steroidal regulator of HPOA aromatase activity in rats.

Estrogen and progestin receptors and aromatase activity in rhesus monkey prostate.

We examined nuclear estrogen receptors (ER) and progestin receptors (PR) in the rhesus monkey prostate. Tissues were obtained from six intact males, five untreated castrates, six castrates treated with testosterone (T) for 6 weeks, and four castrates treated with estradiol (E2) for 6 weeks. Samples of the caudal lobe were either assayed biochemically for ER or stained immunocytochemically (ICC) with monoclonal antibodies against the ER or PR. Prostates from untreated castrates had significantly more ER than tissues from intact or T-treated castrates. In E2-treated castrates, ER number increased compared to that in intact and T-treated castrates. With ICC, ER was found only in the nuclei of the fibroblasts and smooth muscle cells of the stroma, not the glandular, ductal, or urethral epithelial cells. Intact and T-treated castrates had a very small number of positive cells, while untreated and E2-treated castrates had a significantly increased number of positive ER cells in the fibromuscular stroma. With ICC, PR was absent in intact or T-treated animals and barely evident in untreated castrates, but was significantly increased in the fibromuscular stroma of E2-treated castrates. The histological preparations indicated there was no stromal hypertrophy in the E2-treated castrates, but the E2 treatment did cause dilation of the glandular acini. Aromatase activity was measured in prostatic microsomes with a radiometric assay. Levels were low (3-30 fmol/h.mg protein) compared to those in brain and placenta, and no differences in activity were seen between castrates and T-treated castrates. Our data demonstrate that androgens can suppress the level of nuclear ER in the rhesus prostate, and that E2 treatment of castrates can induce PR in the same cells as those that contain ER. Thus, under appropriate conditions, estrogens could affect the rhesus prostate through a receptor-mediated pathway.