Charles E. Roselli

Aromatase activity in the rat brain: Hormonal regulation and sex differences

The intracellular conversion of testosterone to estradiol by the aromatase enzyme complex is an important step in many of the central actions of testosterone. In rats, estrogen given alone, or in combination with dihydrotestosterone, mimics most of the behavioral effects of testosterone, whereas treatment with antiestrogens or aromatase inhibitors block facilitation of copulatory behavior by testosterone. We used a highly sensitive in vitro radiometric assay to analyze the distribution and regulation of brain aromatase activity. Studies using micropunch dissections revealed that the highest levels of aromatase activity are found in an interconnected group of sexually dimorphic nuclei which constitutes a neural circuit important in the control of male sexual behavior. Androgen regulated aromatase activity in many diencephalic nuclei, including the medial preoptic nucleus, but not in the medial and cortical nuclei of the amygdala. Additional genetic evidence for both androgen-dependent and -independent control of brain AA was obtained by studies of androgen-insensitive testicular-feminized rats. These observations suggest that critical differences in enzyme responsiveness are present in different brain areas. Within several nuclei, sex differences in aromatase induction correlated with differences in nuclear androgen receptor concentrations suggesting that neural responsiveness to testosterone is sexually differentiated. Estradiol and dihydrotestosterone acted synergistically to regulate aromatase activity in the preoptic area. In addition, time-course studies showed that estrogen treatment increased the duration of nuclear androgen receptor occupation in the preoptic area of male rats treated with dihydrotestosterone. These results suggest possible ways that estrogens and androgens may interact at the cellular level to regulate neural function and behavior.

Brain aromatization: Classic roles and new perspectives

Aromatization of testosterone to estradiol by neural tissue has classically been associated with the regulation of sexual differentiation, gonadotropin secretion, and copulatory behavior. However, new data indicate that the capacity for aromatization is not restricted to the endocrine brain and demonstrate roles for locally formed estrogens in neurogenesis and in responses of brain tissue to injury. This article summaries our current understanding of the distribution and regulation of aromatase in the brain and describes the classic and novel roles it plays. A better understanding of brain aromatization could shed new light on its physiologic and pathologic functions and someday lead to new, centrally acting drug therapies.

17β-Estradiol rapidly facilitates chemoinvestigation and mounting in castrated male rats

Testosterone and estradiol act synergistically to stimulate male sexual behavior. Previous studies demonstrated that testosterones actions are mediated genomically. Attempts to show that estradiol acts in a similar fashion have been inconclusive. However, estrogens have been shown to exert short-latency effects by acting directly on neuronal membranes. The present experiment examined whether testosterone or estradiol rapidly facilitates copulatory behaviors in castrated sexually experienced rats. Within 35 min of administration, estradiol stimulated chemoinvestigation and frequency of mounting and reduced mount latency in a dose-dependent manner. In contrast, acute administration of testosterone did not alter sexual activity. These data demonstrate for the first time that estradiol exerts short-latency effects on copulatory behavior, providing indirect evidence that this action is mediated through a nontranscriptional mechanism.

Role of P450 aromatase in sex-specific astrocytic cell death

Female animals are protected from ischemic brain damage relative to age-matched males, in part through protection provided by endogenous estradiol. In brain, estradiol is produced from testosterone by cytochrome P450 aromatase (cyp 19), a steroid synthetic enzyme present in astrocytes. We tested the hypothesis that astrocytes derived from neonatal female rat brain are less susceptible than male cells to oxygen–glucose deprivation (OGD), and that this endogenous protection is associated with enhanced aromatase activity. Primary cultured cortical astrocytes were prepared from male and female rat pups separately and grown to confluence in estrogen-free media. Cell death in response to OGD, alone or in combination with hydrogen peroxide, lipopolysaccharides, interleukin-1β, tissue necrosis factor-α, or nitric oxide (NO) donor diethylenetriamine/nitric oxide adduct (DETA/NO) was quantified as the ratio of propidium iodide to calcein AM-positive cells. Aromatase activity and cyp19 mRNA and protein levels were measured in cultures from each sex. Female astrocytes are more resistant to OGD and oxidant cell death induced by H2O2, but sustain greater cell death when inflammatory mediators are combined with OGD compared with OGD alone. Media transfer from female to male cells conferred protection against OGD-induced cell death. Aromatase activity and expression is greater in female than in male astrocytes. The aromatase inhibitor, Arimidex (100 nmol/L), abolishes sex differences in OGD-induced cell death, whereas treatment with 17β-estradiol (10 nmol/L) protects cells of either sex. We conclude that astrocytes isolated from neonatal cortex exhibit marked sex differences in sensitivity to OGD, in part because of enhanced aromatization and estradiol formation in female cells.

PRENATAL HORMONES ORGANIZE SEX DIFFERENCES OF THE NEUROENDOCRINE REPRODUCTIVE SYSTEM : OBSERVATIONS ON GUINEA PIGS AND NONHUMAN PRIMATES

1. The central nervous systems (CNS) of males and females differ in the control mechanisms for the release of gonadotropins from the anterior pituitary gland as well as the capacity to display sex specific behaviors. 2. In guinea pigs and monkeys, these differences are organized through the actions of prenatal androgens secreted by the fetal testes. In both males and females androgen receptors have been identified within the brain during the period in development in which organization of the CNS occurs. Sex differences between the ratio of cytosolic and nuclear androgen receptors are due to the amount of endogenous androgen present in the circulation of the developing fetus. Thus, at least part of the biochemical machinery necessary for androgen action resides in the CNS during the period of sexual differentiation. 3. In addition to the physiological differences that have been observed, morphological differences that are androgen dependent have been found in the medial preoptic nucleus and the bed nucleus of the stria terminalis of the guinea pig. The location of these sex differences in brain morphology coincides roughly with the location of steroid binding neurons. 4. In some species the in situ conversion of testosterone to dihydrotestosterone (DHT) by the 5 alpha-reductases or to estradiol-17 beta by cytochrome P450 aromatase mediates testosterones action. The gonadotropin surge mechanism of adult guinea pigs exposed to a 5a-reductase inhibitor in utero during the critical period for sexual differentiation was unaffected in either males or females even though the development of the external organs of reproduction of males was feminized by the treatment. Likewise, the gonadotropin surge mechanism of subjects exposed to an aromatase inhibitor in utero during the critical period for sexual differentiation was unaffected by this treatment. 5. The mechanism controlling negative feedback, however, was affected in both males and females. Subjects that were exposed to an aromatase inhibitor while developing in utero could not respond to the negative feedback actions of estrogen on gonadotropin release in adulthood. 6. The surge mechanism for the control of gonadotropin secretion in nonhuman primates is not sexually differentiated as it is in rodents. Castrated male monkeys release surge amounts of LH in response to an estrogen challenge. Both infant and adult dimorphic behaviors of rhesus monkeys are organized by the prenatal actions of androgen.

Anatomic relationships between aromatase and androgen receptor mRNA expression in the hypothalamus and amygdala of adult male cynomolgus monkeys

This study mapped the regional locations of cells expressing cytochrome P450 aromatase (P450AROM) and androgen receptor (AR) mRNAs in the adult male macaque hypothalamus and amygdala by in situ hybridization histochemistry using monkey‐specific cRNA probes. High densities of P450AROM and AR mRNA‐containing neurons were observed in discrete hypothalamic areas involved in the regulation of gonadotropin secretion and reproductive behavior. P450AROM mRNA‐containing neurons were most abundant in the medial preoptic nucleus, bed nucleus of the stria terminalis, and anterior hypothalamic area, whereas AR mRNA‐containing neurons were most numerous in the ventromedial nucleus, arcuate nucleus, and tuberomamillary nucleus. Moderate to heavily labeled P450AROM mRNA‐containing cells were present in the cortical and medial amygdaloid nuclei, which are known to have strong reciprocal inputs with the hypothalamus. Heavily labeled P450AROM mRNA‐containing cells were found in the accessory basal amygdala nucleus, which projects to the cingulate cortex and hippocampus, areas that are important in the expression of emotional behaviors and memory processing. In contrast to P450AROM, the highest density of AR mRNA labeling in the temporal lobe was associated with the cortical amygdaloid nucleus and the pyramidal cells of the hippocampus. All areas that contained P450AROM mRNA‐expressing cells also contained AR mRNA‐expressing cells, but there were areas in which AR mRNA was expressed but not P450AROM mRNA. The apparent relative differences in the expression of P450AROM and AR mRNA‐containing neurons within the monkey brain suggests that T acts through different signaling pathways in specific brain areas or within different cells from the same region. J. Comp. Neurol. 439:208–223, 2001.

Quantitative Distribution of Nuclear Androgen Receptors in Microdissected Areas of the Rat Brain

The binding of androgens to specific high-affinity receptor sites in brain tissue is postulated as an initial event in the mechanism of central androgenic action. In an effort to assess the functional capacity of the androgen receptor system in the central nervous system, we measured the concentration of nuclear (ARn) as well as cytosolic androgen receptors (ARc) in 13 microdissected brain samples from intact male and female Sprague-Dawley rats. Tissues from 6 rats were combined for each determination and androgen receptor contents were measured with single-point in vitro assays that used saturating concentrations of high specific activity 3[H]dihydrotestosterone. We found that ARc levels tended to be higher in females than males although the general patterns of distribution were very similar. As expected, ARn concentrations were significantly higher in males than females. The highest concentrations of ARn (greater than 100 fmol/mg DNA) in males were measured in the ventromedial nucleus of the hypothalamus and medial amygdala; intermediate levels (50-100 fmol/mg DNA) were found in arcuate nucleus-median eminence, medial preoptic nucleus, periventricular preoptic area, bed nucleus of the stria terminalis, anterior hypothalamus, periventricular anterior hypothalamus, lateral septum, and parietal cortex, and low levels (less than 50 fmol/mg DNA) were measured in lateral preoptic nucleus and cortical amygdala. With the exception of the periventricular preoptic area (74 +/- 33 fmol/mg DNA), only very low concentrations of ARn were measured in females. These data provide the first quantitative profile of ARn in discrete brain nuclei and subregions of the rat.

Synergistic Induction of Aromatase Activity in the Rat Brain by Estradiol and 5α-Dihydrotestosterone

Estrogens are produced locally from androgen precursors by cells within the hypothalamus and preoptic area (POA). The activity of the aromatase enzyme complex responsible for this intracellular conversion is controlled by gonadal steroids. The purpose of this study was to determine: (1) whether estrogen acts together with androgen to regulate aromatase activity (AA) and (2) whether nuclear androgen receptor levels are increased after exposure to combined treatment with estradiol benzoate (EB) and dihydrotestosterone (DHT). Thus, adult male rats were castrated and treated for 1 week with either vehicle (0.1 ml sesame oil, s.c.), EB (2 micrograms/day), testosterone (3-cm Silastic implant), DHT (3-cm Silastic implant) or EB + DHT. These treatments produced hormone levels in the physiologic range. We found that both testosterone and DHT significantly stimulated AA (p less than 0.05 vs. castrated rats). However, testosterone induced AA significantly more than DHT in the POA (p less than 0.05; castrated + testosterone vs. castrated + DHT). EB alone did not affect AA but synergized with DHT to stimulate AA in the POA to levels equivalent with the testosterone-treated group. By comparison, EB alone did not enhance the induction of AA by DHT in the hypothalamus. Combined treatment with EB and DHT had no effect on the concentrations of nuclear androgen receptors in either tissue suggesting that the effect of EB was not mediated through an androgen receptor mechanism. These results suggest that both androgens and estrogens play a physiologic role in the control of estrogen formation in the rat brain. Furthermore, the anatomical specificity that we observed indicates that critical differences in enzyme responsiveness are present in different areas of the brain.

Sex differences in androgen-regulated expression of cytochrome P450 aromatase in the rat brain ☆

The basis of functional gender differences in adult responsiveness to testosterone (T) is not yet understood. Conversion of T to estradiol by cytochrome P450 aromatase in the medial preoptic area is required for the full expression of male sexual behavior in rats. High levels of aromatase are found in the medial preoptic nucleus (MPN) and in an interconnected group of sexually dimorphic nuclei which mediate masculine sexual behavior. Within this neural circuit, aromatase is regulated by T, acting through an androgen receptor (AR)-mediated mechanism. This arrangement constitutes a feedforward system because T is both the regulator and the major substrate of aromatase. Preoptic aromatase is thus more active in adult males than in females because of normal sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic because equivalent physiological doses of T stimulate aromatase to a greater extent in males than in females. Dose-response studies indicate that the sex difference is apparent over a range of circulating T concentrations and constitute a gender difference in T efficacy, but not potency. Sex differences in aromatase correlate with sex differences in nuclear AR concentrations in most regions of the sexually dimorphic neural circuit, but not in MPN. These results suggest that males may have larger populations of target cells in which aromatase is regulated by androgen, but the lack of a gender difference in AR levels in the MPN suggests that differences in post-receptor mechanisms could also be involved. Measurements of aromatase mRNA in androgen-treated gonadectomized rats demonstrate that sex difference in regulation is exerted pretranslationally. Taken together these results demonstrate a sexually dimorphic mechanism that could potentially limit the action of T in females, and may relate to the enhanced expression of T-stimulated sexual behaviors in males.

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.