John A. Resko

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.

Plasma testosterone and male dominance in a Japanese macaque (Macaca fuscata) troop compared with repeated measures of testosterone in laboratory males

Abstract Plasma testosterone levels measured by radioimmunoassay did not correlate with dominance rank or aggressive behavior in 21 adult males of a natural troop of Japanese macaques (Macaca fuscata). The data were replicated during two consecutive breeding seasons. Levels of male dominance and aggressive behavior were highly correlated from year to year, but testosterone levels of individual males were not. Nor were individual levels of testosterone correlated in five laboratory males sampled every 15 min over a 2-hr period. These laboratory males showed multiple, apparently random peaks but the mean testosterone level of 11.73 ng/ml did not vary significantly throughout the 120 min that were studied. By demonstrating that the stress of blood collection did not depress plasma testosterone titers in laboratory males, these data validated the method used in the study of the natural troop.

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.

A new antiserum with conformational specificity for LHRH: Usefulness for radioimmunoassay and immunocytochemistry

We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.

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.

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.

The distribution and regulation of aromatase activity in the central nervous system.

Our data demonstrate that androgen-dependent AA is found in areas of the brain that are essential for the neuroendocrine control of gonadotropin secretion and sexual behavior. However, until we know more about the neurons that contain AA, e.g., whether they are peptidergic or catecholaminergic, we can not speculate about the neuronal functions that depend on local estrogen formation. In fact, the association of AA with neurons and not glia has only recently been demonstrated. That estrogens and androgens synergize in the regulation of various neuroendocrine functions has been known for many years, but an explanation of the synergism at the cellular level was not available. One explanation for this synergism may lie in our recent observation that the administration of exogenous estradiol to castrated rats increases androgen-receptor concentrations in specific brain nuclei. Perhaps locally formed estrogens work in a similar fashion to regulate androgen receptors in the brain of the intact male.

Cytochrome P450 aromatase in testis and epididymis of male rhesus monkeys

To understand the role of estrogen in testicular and epididymal function of rhesus monkeys, we measured steroids in the spermatic and peripheral venus circulation and aromatase activity and its mRNA in testis and epididymis. Testosterone, estradiol-17β, and estrone, but not androstenedione, were elevated in the spermatic vein serum compared to the peripheral circulation. Aromatase activity in testis and in caput epididymis (259±16 [SEM] vs 274±47 fmol of 3H2O/mg of protein/h [n=10], respectively) was significantly higher (p<0.01) than in corpus and cauda (124±28 and 113±33 fmol of 3H2O/mg of protein/h [n=10], respectively). In the ribonuclease protection assay, two P450arom mRNA transcripts were identified in testis and epididymis. One corresponded with the aromatase full-length transcript and the other was a truncated isoform. The latter was significantly more abundant than the former (p<0.01). Our results demonstrate that the monkey testis and, to a lesser extent, the epididymis can aromatize androgens. However, in the epididymis, like in some areas of the brain, there was a discrepancy between the aromatase activity and the mRNA. The fact that P450arom mRNA and aromatase activity do not correlate in the epididymis may indicate that aromatase activity is not strictly regulated at the level of RNA expression and that other mechanisms for this regulation should be considered.