Region-specific regulation of cytochrome P450 aromatase messenger ribonucleic acid by androgen in brains of male rhesus monkeys

We demonstrated previously that testosterone regulates aromatase activity in the anterior/dorsolateral hypothalamus of male rhesus macaques. To determine the level of the androgen effect, we developed a ribonuclease protection assay to study the effects of testosterone or dihydrotestosterone (DHT) on aromatase (P450(AROM)) mRNA in selected brain areas. Adult male rhesus monkeys were treated with testosterone or DHT. Steroids in serum were quantified by RIA. Fourteen brain regions were analyzed for P450(AROM) mRNA. Significant elevations of its message over controls (P<0.05) were found in the medial preoptic area/anterior hypothalamus of both androgen treatment groups and the medial basal hypothalamus of the testosterone-treated males. Other brain areas were not affected by androgen treatment. We conclude that testosterone and DHT regulate P450(AROM) mRNA in brain regions that mediate reproductive behaviors and gonadotropin release. The P450(AROM) mRNA of other brain areas is not androgen dependent. Brain-derived estrogens may also be important for maintaining neural circuitry in brain areas not related to reproduction. The control of P450(AROM) mRNA in these areas may differ from what we report here, but it is equally important to understand the function of in situ estrogen formation in these areas.     

Hypothalamic control of the post-castration rise in serum LH concentration in rams

Sexually mature rams were left intact, castrated (wethers), castrated and implanted with testosterone, or castrated, implanted with testosterone and pulse-infused every hour with LHRH. Serum concentrations of LH increased rapidly during the first week after castration and at 14 days had reached values of 13.1 +/- 2.2 ng/ml (mean +/- s.e.m.) and were characterized by a rhythmic, pulsatile pattern of secretion (1.6 +/- 0.1 pulses/h). Testosterone prevented the post-castration rise in serum LH in wethers (1.0 +/- 0.5 ng/ml; 0 pulses/h), but a castrate-type secretory pattern of LH was obtained when LHRH and testosterone were administered concurrently (10.7 +/- 0.8 ng/ml; 1.0 pulse/h). We conclude that the hypothalamus (rather than the pituitary) is a principal site for the negative feedback of androgen in rams and that an increased frequency of LHRH discharge into the hypothalamo-hypophysial portal system contributes significantly to the post-castration rise in serum LH.     

Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance

In adult male primates, estrogens play a role in both gonadotropin feedback and sexual behavior. Inhibition of aromatization in intact male monkeys acutely elevates serum levels of luteinizing hormone, an effect mediated, at least partially, within the brain. High levels of aromatase (CYP19) are present in the monkey brain and regulated by androgens in regions thought to be involved in the central regulation of reproduction. Androgens regulate aromatase pretranslationally and androgen receptor activation is correlated with the induction of aromatase activity. Aromatase and androgen receptor mRNAs display both unique and overlapping distributions within the hypothalamus and limbic system suggesting that androgens and androgen-derived estrogens regulate complimentary and interacting genes within many neural networks. Long-term castrated monkeys, like men, exhibit an estrogen-dependent neural deficit that could be an underlying cause of the insensitivity to testosterone that develops in states of chronic androgen deficiency. Future studies of in situ estrogen formation in brain in the primate model are important for understanding the importance of aromatase not only for reproduction, but also for neural functions such as memory and cognition that appear to be modulated by estrogens.     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.     

Neonatal castration of male and female rats affects luteinizing hormone responses to estrogen during adulthood

We examined the positive and negative feedback effects of estradiol (E2) on luteinizing hormone (LH) and prolactin (Prl) secretion in adult male and female rats which were gonadectomized within 24 h after birth (long-term castrates) and compared these responses to those elicited by E2 in short-term castrated (7 days) adult males and females. The high serum E2 did not reduce the elevated serum LH concentrations in long-term castrates until 4 days of treatment. Also, only after negative feedback was established were the positive feedback actions of E2 observed. In contrast, Prl surges were observed after 2 days of E2, and baseline Prl serum levels were elevated by Day 3 of E2 in long-term castrated male and female rats. Some long-term castrates lacked both LH and Prl surges, and E2 was ineffective in altering basal gonadotropin secretion in these animals. Short-term castrated males had elevated serum Prl levels but no Prl surges. Seemingly, when the hypothalamus is deprived of estrogen or androgen from birth to adulthood, an equal percentage of males and females become refractory to the positive feedback effects of estrogen during adulthood. Thus, it is difficult to separate castration effects from those which may be produced by the endogenous androgen secreted during the first 26 h of life.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Effects of alcohol feeding on androgen receptors in the rat pituitary gland

Specific binding of testosterone-1 beta, 2 beta-3H by cytosol from anterior pituitary gland of alcohol-fed, isocaloric control, and castrated control and alcohol-fed rats with or without testosterone treatment has been investigated by charcoal assay. The number of androgen binding sites was significantly reduced in alcohol-fed rats (8 +/- 1.0 fmoles/mg cytosol protein), when compared to the isocaloric control value (13.2 +/- 2.1 fmoles/mg protein), with no significant change in Kd (0.7 +/- 0.14 nM). Castration significantly increased the number of receptor sites in control rats and when castrated control animals were treated with testosterone the binding sites were decreased to the intact control level. In contrast, castration or testosterone given to castrated alcohol-fed rats did not alter alcohol-induced reduction of the receptor sites. The binding affinity (Kd) is identical in all groups. The concentration of serum luteinizing hormone (LH) was significantly lower in alcohol-fed rats when compared to that of normal controls. An increased serum LH level with a decreased testosterone level was noted in castrated control rats. However, castration of alcohol-fed rats had little or no effects on the concentrations of LH and testosterone. Administration of testosterone suppressed castration-induced high LH in control rats but alcohol-induced reduction of LH level was not altered by this treatment. These findings indicate that alcohol exerts a suppressive effect on the content of androgen receptors and secretory functions of gonadotropins in the pituitary gland.     

Brain aromatase in control versus castrated Norway Brown, Sprague-Dawley and Wistar adult rats.

Brain aromatase cytochrome P450 converts androgens to estrogens that play a critical role in the development of sexually dimorphic neural structures, the modulation of neuroendocrine function(s), and the regulation of sexual behavior. We characterized the influence of surgical castration on brain aromatase in Norway Brown and Wistar adult rats and compared their responses to Sprague-Dawley rats that were surgically or biochemically castrated (with flutamide, a known androgen receptor blocker). Aromata enzyme activity was measured by the tritiated water release assay in the medial basal hypothalmus/preoptic area (MBH/POA) and amygdala brain regions. The present results demonstrate that independent of the rat strain examined, MBH/POA aromatase is regulated by androgens (in Sprague-Dawley, Norway Brown and Wistar males). However, intact Wistar animals displayed significantly higher MBH/POA aromatase levels compared to Sprague-Dawley control values. Conversely, in the amygdala region, there was an apparent lack of androgen hormone action upon aromatase enzyme activity in some of the rat strains tested. The importance of brain aromatase regulating estrogen biosynthesis and influencing brain development and function is covered.     

The subcellular localization of the changes in ribonuclease and phosphatase activities of the mouse kidney produced by castration and testosterone

Male mice were castrated at 2 months of age and a pellet of testosterone propionate was implanted subcutaneously ten days later. After 14 days, normal, castrated and androgen treated mice were autopsied simultaneously. The kidneys of each group were homogenized, pooled and fractionated by centrifugation into the various subcellular components. The alkaline ribonuclease and phosphatase activities were localized in the microsomal fraction and changed in inverse relationship to the previously observed changes in the rate of protein biosynthesis and the concentration of microsomal and polysomal RNA induced by castration and androgen administration. Esterase activity also was localized in the microsomal fraction and its specific activity decreased after castration and was restored to normal by testosterone. The activities of acid ribonuclease, acid phosphatase and the several marker enzymes (glucose-6-phosphatase, nucleotidase, succinic dehydrogenase and glucose-6-phosphate dehydrogenase) changed in direct proportion to the changes in weight of the kidney after castration and androgen stimulation.     

A 'window of time' during which testosterone determines the opiatergic control of LH release in the adult male rat

Male rats castrated before puberty (when 26 days of age) showed a progressively decreasing susceptibility to the inhibitory effects of morphine (5 mg/kg) upon LH secretion for up to 28 days after gonadectomy (approximately 100%, 40% and 10% inhibition at 5, 12 and 28 days after castration), but thereafter morphine again caused approximately 50% reduction in serum LH values; the minimum inhibition found at 28 days after castration (age 54 days) occurred at the time at which male rats normally reach puberty. When rats were castrated at 59 days of age, morphine maximally suppressed serum LH concentrations (to less than 70%) 2 and 5 days after castration, but had no effect thereafter. In prepubertal castrates, testosterone replacement between Days 26 and 50 of life resulted in responses to morphine similar to those found in rats castrated after puberty, i.e. serum LH levels were not reduced. Morphine significantly reduced LH levels in prepubertal castrates given testosterone after 60 days of age. Treatment with morphine consistently elevated serum prolactin concentrations (greater than 100%) in castrated rats of all ages, regardless of the time elapsed after gonadectomy. These results indicate a transient fall in the inhibitory opioidergic tone upon LH secretion as the normal age of puberty approaches, that the ability of opiates to alter LH release in adulthood may depend upon testicular steroids secreted during the peripubertal period, and that the LH responses do not reflect general changes in the neuroendocrine response to opiates after castration since the prolactin response to morphine remains intact in rats castrated before and after puberty.     

Effects of adrenocorticotrophin (ACTH) and progesterone on luteinising hormone (LH) secretion in recently castrated rams.

The goal of the present study was to determine whether ACTH and progesterone have any effect on LH secretion and pulse frequency in recently castrated rams. Six 2-year-old Corriedale rams were castrated in the winter. The day before castration, blood samples were taken in order to establish the precastration LH levels. The rams were divided into an untreated group (group U: n = 2) and a treated group (group T: n = 4). The first treatment consisted of the i.v. administration of 0.5 mg of ACTH on day 20 post-castration, immediately after the first sample had been taken. During the second treatment, subcutaneous progesterone implants were given to group T for 5 days. Control samplings were performed one week before each treatment. Prior to castration, the testosterone levels were low, while after castration they were below the detection limit of the assay. Cortisol and progesterone concentrations were basal before castration in all of the animals and after castration in group U and also in the control samplings for group T. ACTH treatment caused a significant increase in both cortisol and progesterone levels for 3 h (P < 0.001). Progesterone implants raised progesterone levels in group T, but cortisol levels remained basal. Before castration, all animals had low LH levels and hardly any pulse activity was seen. After castration, both the number of LH pulses and the mean LH production increased significantly in all of the animals (P < 0.01). During the ACTH trial, LH pulse frequency was significantly reduced for the first 4 h following ACTH administration (P = 0.013), however, no such differences occurred in the prior control period. No effect was seen on mean LH concentration during the ACTH treatment. Progesterone treatment did not have any effect on either the number of LH pulses nor on LH concentrations (P > 0.05).     

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Effects of androgen administered to neonatal male rats on hypophyseal gonadotropin content, secretion and response to LHRH at adulthood

Supraphysiologic doses (1.75-3.50 mg) of testosterone propionate (TP) administered to male rats on the day of birth and 24 h later resulted in markedly reduced serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in adult males castrated for 16 days. These effects diminished as androgen was injected on succeeding postnatal days. Since exogenous dihydrotestosterone and testosterone were similarly effective, aromatization to estrogen is not required to elicit these effects. No build-up of either gonadotropin occurred in the pituitaries of TP-treated animals; pituitary LH content was appreciably reduced, while FSH remained unchanged. These data imply that hypophyseal synthesis and secretion of gonadotropins are curtailed in adult castrated males who have been androgenized neonatally. Pituitaries of such neonatally treated animals, however, were capable of increased secretion of LH in response to a challenge of luteinizing hormone-releasing hormone. These findings are compatible with a model in which an androgen suppressible event occurs at a suprahypophyseal level, e.g., hypothalamus or higher brain centers, in the male rat during a restricted neonatal period, which is responsible for programming the development of mechanisms involved in accumulation and secretion of gonadotropins.     

Phenotype characteristics of transgenic male mice expressing human aromatase under ubiquitin C promoter

To study the significance of the increased ratio of the estrogen/androgen concentration for the male reproductive functions, we have generated transgenic mice expressing human P450 aromatase under a promoter providing ubiquitous and permanent transgene expression (AROM+ mice). AROM+ male mice are characterized by elevated serum estradiol and prolactin (Prl) concentrations, combined with markedly reduced testosterone levels. The mice are present with a multitude of structural and functional alterations in the reproductive organs such as cryptorchidism, Leydig cell hyperplasia, disrupted spermatogenesis and infertility. Furthermore, the mice develop infravesical obstruction associated with the rhabdosphincter atrophy and rudimentary accessory sex glands. Interestingly, the mammary gland in AROM+ males undergo a ductal and alveolar development morphologically resembling terminally differentiated female mammary glands, and express several signaling proteins typical for female mammary glands. Some of the abnormalities seen in AROM+ mice are similar to those described in both mice and humans exposed to diethylstilbestrol (DES) in utero. The importance of the AROM+ model may lie in its predictability, i.e. the model suggests which abnormalities of the human reproductive functions may be associated with the increased ratio of estrogen/androgen concentrations in early life and at adult age as well.     

Testosterone regulates aromatase activity in discrete brain areas of male rhesus macaques

The nonhuman primate brain contains two divergent pathways for testosterone (T) metabolism. Estradiol is biosynthesized from T by aromatization through the first pathway, whereas dihydrotestosterone is produced by the action of 5 alpha-reductase through the second pathway. Previously, we mapped the distribution of these enzyme activities within specific microdissected brain area and determined that aromatase activity (AA), but not 5 alpha-reductase activity (5 alpha RA), was reduced in certain brain areas after castration. In the present study, we measured AA and 5 alpha RA in thirteen brain nuclei and subregions from five castrated and five T-treated castrated male rhesus monkeys to determine whether exogenous androgen treatment could reverse the effects of castration on brain AA. We found that T, administered in a dose that maintained serum levels at 14.2 +/- 1.6 (SEM) ng/ml, suppressed circulating luteinizing hormone (Castrates = 491.9 +/- 86 ng/ml vs. T-treated castrates = 1.8 +/- 0.2 ng/ml), and stimulated AA in specific nuclei including the suprachiasmatic nucleus (n.), periventricular area, ventromedial n., and lateral hypothalamus. T treatment had no significant effect on AA in nine other nuclei or on 5 alpha RA in any brain areas that we studied. These data indicate that AA in diencephalic and limbic structures of the nonhuman primate brain is distributed heterogeneously into androgen-dependent and androgen-independent regions. This distribution is similar to that found in rodents. 5 alpha RA, on the other hand, is more homogeneously distributed than AA in these same brain regions and is not controlled by androgens.     

Influence of perinatal androgenization on the castration response of adult rats

An unexplained dichotomy exists between the LH (luteinizing hormone) responses to castration of male and female rats, as males show a more prompt increase in serum LH levels. We have tested the hypothesis that neonatal exposure to androgen determines the sexual dimorphism of that response. Control groups of male and female rats were castrated at 60 days of age. Other animals had been castrated at 0 or 25 days of age and then given steroid treatment via testosterone (T) implants from 25 through 60 days of age. At 60 days of age a blood sample was taken from each animal before removal of either the T implant or the gonads. Animals were bled again 24 and 48 h later. Within 24 h after orchidectomy the typical early plateau of plasma LH had occurred, represented by an increment in mean LH concentrations of 316 ng/ml. Orchidectomy at 25 days of age had little or no effect on subsequent response to removal of T. In contrast, neonatal orchidectomy resulted in a markedly diminished response to T removal on Day 60. The response, however, was not reduced to that of normal females. In female rats plasma LH does not increase by 48 h after ovariectomy. Perinatal testosterone propionate (TP) treatment of females partially masculinized (enhanced) the LH response to T implant removal, but only if ovariectomy had been performed prior to puberty (at 0 or 25 days of age).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of short days on aromatization and accumulation of nuclear estrogen receptors in the hamster brain

Exposure of hamsters to short days increases sensitivity to the negative feedback effects of testosterone (T) but decreases responsiveness to the behavioral effects of the hormone. Since T is metabolized in the brain to 5 alpha-dihydrotestosterone (DHT) and estradiol, which differentially affect gonadotropin secretion and sex behavior, it is reasonable to postulate that daylength can modulate neural responses by quantitative or qualitative alterations in T metabolism and subsequent receptor binding of active hormone. Experiments reported here focused on aromatization and the nuclear accumulation of estrogen receptors. Adult male hamsters were maintained for 6-12 wk in long (14:10 LD) or short (8:16 LD) daily photoperiods. Both intact and castrated animals were used to assess direct effects of short days versus changes due to short-day-induced testicular regression. Discretely dissected regions of the brain (preoptic area, POA; hypothalamus, HTH; and corticomedial amygdala, CMA) or limbic blocks (LIM) comprised of all three regions were assayed for estrogen-synthesizing activity (aromatase) and estrogen-binding activity (receptors). Aromatase was estimated in vitro by conversion of [7-(3)H] androstenedione to [3H] estrogen and in vivo by measuring increases in nuclear estrogen receptor levels after injection of aromatizable androgen. Receptor-binding activity was assayed in crude cytosolic and nuclear extracts by incubating samples with [3H] estradiol +/- 100-fold excess inert estradiol, and separating free and bound steroids by Sephadex LH-20 gel filtration. When aromatase was assayed in homogenates prepared from discrete brain regions of individual hamsters, significantly lower activity was found in the HTH of short-day animals than in long-day controls. This effect was seen in both intact and castrated animals, which indicates that it was not mediated by the testis. Decreased enzyme activity in the POA and CMA of short-day hamsters was not significant, nor was there an effect of castration independent of short days. Low levels of nuclear estrogen receptors were present in LIM of intact males, but these were reduced after castration or concomitant with testicular regression after short-day exposure. This suggests that the hamster testis normally secretes estrogen or aromatizable androgen. A single injection of estradiol or aromatizable androgen (T or androstenedione) increased nuclear receptors in LIM of castrated animals. Cytosolic receptors were not different in short-day vs. long-day hamsters, nor were there differences in nuclear receptor levels after a single estradiol injection.(ABSTRACT TRUNCATED AT 400 WORDS)