Biomedical subjects

C E Roselli

Publications and source records attributed to C E Roselli.

At least 37 records · Page 2Linked to original sources

PubMed1998

Anatomic distribution and regulation of aromatase gene expression in the rat brain.

Recent evidence suggests that two cytochrome P450 aromatase (P450arom) mRNA transcripts are present in the rat brain. One of these contains the entire 5'-coding sequence and correlates with the presence of functional enzyme. We designed a new 255-base pair P450arom probe (AROM255) that recognizes only this full-length P450arom mRNA. Ribonuclease protection assays verified that the cRNA probe synthesized from this construct recognized a single RNA species in brain tissues that express aromatase activity, but not in the cingulate cortex, an area previously shown to contain only the alternate transcript. Moreover, the P450arom mRNA content of the preoptic area was significantly lower in castrates than in intact males or testosterone (T)-treated castrates. We employed 33P-labeled cRNA probes to examine the distribution of P450arom mRNA by in situ hybridization. High levels of mRNA were detected in the medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BnST), and medial amygdala (MA). Lower levels were found in the ventromedial hypothalamic nucleus and cortical amygdala. The magnitude of the hybridization signal in the BnST and MPN was greater in males than in females. Treatment with T propionate significantly increased hybridization signal in BnST, MPN, and MA. These results confirm the anatomic distribution of P450arom mRNA within hypothalamic and limbic nuclei of the adult male rat and demonstrate that steady state concentrations are regionally regulated by T. Moreover, they demonstrate the necessity of using a molecular probe that can distinguish between P450arom variants in the brain.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

Expression of the orphan receptor steroidogenic factor-1 mRNA in the rat medial basal hypothalamus.

Steroidogenic factor-1 (SF-1), an orphan receptor of the nuclear hormone receptor family, binds to the AAGGTCA motif in the promoter elements of several diverse target genes, including some that mediate steroidogenesis and sexual differentiation. In addition, SF-1 is expressed in embryonic forebrain, suggesting that it plays a role in neural development. This study was undertaken to study the distribution and regulation of SF-1 mRNA expression in the rat brain. SF-1 mRNA levels were measured in tissue dissections by ribonuclease protection assay. A 452 nt 32P-labeled cRNA probe, complementary to the putative ligand-binding domain of the rat SF-1 mRNA, was synthesized from the rat SF-1 cDNA inserted into pBluescript II KS, using a Sty 1 fragment and T3 polymerase. The probe protected a single 390 nt transcript in the medial basal hypothalamus (MBH) and peripheral steroidogenic tissues of the male rat. The size of this protected band corresponded to that of the protected sense RNA standard (HindIII fragment of the SF-1 cDNA transcribed with T7 polymerase). No SF-1 mRNA was detected in the preoptic area, amygdala or cingulate cortex. The levels of SF-1 mRNA in MBH were not affected by gonadectomy or androgen treatment, nor was there a sex difference in its expression in adults. In situ hybridization histochemistry revealed that SF-1 was localized to the ventromedial nucleus of the adult hypothalamus. The levels of SF-1 mRNA were high on gestational day 18 after which they fell by approximately 30% and remained constant throughout gestation, the first week of neonatal life, and into adulthood. These results demonstrate that the gene encoding SF-1 is expressed in a discrete region of the rat hypothalamus and appears to be developmentally regulated, but not affected by gonadal hormones in adults.

Age Factors↗

PubMed1997

Regulation of aromatase gene expression in the adult rat brain.

Brain aromatase plays an important role in the regulation of adult reproductive behavior in male rodents. This report focuses on recent experiments from our laboratory that examined the distribution and regulation of aromatase mRNA in the rat brain. Aromatase mRNA was measured by a highly sensitive ribonuclease protection assay using a 32P-labeled antisense RNA probe that was complimentary to the 5' coding region of rat aromatase mRNA. This probe protects two RNA fragments in rat brain tissue: a 430-nt length fragment and a shorter 300-nt fragment. The presence of the 300-nt RNA fragment is not associated with enzyme activity in the rat brain and appears to represent an alternative brain-specific aromatase transcript whose function, if any, is unknown. In contrast, the 430-nt RNA fragment represents mRNA, which is thought to encode functional aromatase enzyme because its levels are correlated with aromatase activity concentrations in preoptic area, hypothalamus, amygdala, and ovary. Aromatase activity and mRNA levels in the preoptic area and hypothalamus decreased by 7 days after castration and were maintained at intact levels by treatment with testosterone and dihyhdrotestosterone, but not with estradiol. In contrast, neither aromatase activity nor mRNA levels in the amygdala are affected by castration or hormone replacement. In addition, sex differences in the regulation of aromatase mRNA were apparent in both the preoptic area and hypothalamus. These results demonstrate that androgens regulate the transcription or stability of aromatase mRNA in specific brain areas. Moreover, they suggest that gender differences in androgen responsiveness play an important role in regulating gene expression in the adult rat brain.

Alternative Splicing↗

PubMed1997

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 testosterone's 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.

Animals↗

PubMed1997

Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains.

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450arom) is an important step in the mechanism of androgen action in the brain. However, the distribution of P450arom mRNA in adult rhesus monkey brains has not been studied because specific probes have not been available. To address this deficit, we cloned and sequenced a 455-basepair segment of the 5' coding region of the rhesus P450arom cDNA. Total RNA was extracted from a rhesus monkey placenta (Day 47 of gestation and subjected to reverse transcriptase (RT) polymerase chain reaction (PCR) using consensus oligonucleotide primers selected from published human and rat P450arom DNA sequences. The RT-PCR product was subcloned into a vector and sequenced. The monkey P450arom cDNA was 97% identical to the human sequence but shared only 86% homology with the rat sequence. We then developed a ribonuclease protection assay using a monkey P450arom cDNA and studied the distribution of P450arom mRNA in adult monkey brains. This assay protected two RNA fragments, one 455 nucleotides (nt) in length and the second approximately 300 nt. The relative distribution of P450arom mRNA (the 455-nt fragment) between brain areas of the adult (n = 3) was high in the bed nucleus of the stria terminalis > medial preoptic/anterior hypothalamus > amygdala; intermediate in the medial basal hypothalamus (infundibular nucleus, median eminence, ventromedial nucleus) > lateral preoptic/anterior hypothalamus; and low in the septum > lateral-dorsal-medial hypothalamus. P450arom mRNA was undetectable in cingulate and parietal cortex, hippocampus, and cerebellum. P450arom activity, as measured by the 3H2O assay, correlated well with the distribution of P450arom mRNA (the 455-nt protected fragment; r = 0.9) in the same tissues. A shorter protected RNA fragment was found in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the amygdala, and the cingulate and parietal cortex but not in the other brain areas investigated. Its presence did not correlate with aromatase activity in brain tissue. This study describes the development of a ribonuclease protection assay using a monkey cDNA produced by RT-PCR and its usefulness for studying the distribution of P450arom mRNA in brains of nonhuman primates.

Animals↗

PubMed1997

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.

Androgens↗

PubMed1996

Levels of serum steroids, aromatase activity, and estrogen receptors in preoptic area, hypothalamus, and amygdala of B6D2F1 male house mice that differ in the display of copulatory behavior after castration.

Most male B6D2F1 hybrid house mice continue to copulate after castration (continuers), whereas others do not (noncontinuers). Copulation in continuers appears estrogen dependent. Serum testosterone (T), 17 beta-estradiol (E2), and dihydrotestosterone (DHT), as well as aromatase activity (AA) and estrogen receptor (ER) levels in preoptic area (POA), hypothalamus (HYP), and amygdala (AM) were measured to determine if continuers and noncontinuers differ in estrogen physiology. In general, continuers and noncontinuers did not differ in serum steroid levels, AA, or ER levels. Castration reduced AA in the POA, HYP, and AM. Castration did not affect nuclear ER levels in the POA and HYP but reduced nuclear ER in AM. The data demonstrate that castrated B6D2F1 male mice continue to be under the influence of circulating nongonadal E2 that is important for copulation.

Amygdala↗

PubMed1996

Effect of vorozole, an aromatase enzyme inhibitor, on sexual behavior, aromatase activity and neural immunoreactivity.

Aromatase enzyme is essential for the expression of normal sexual behavior in many mammals and birds. Here we report that vorozole (R83842), a non-steroidal aromatase inhibitor, blocks sexual behavior in the female musk shrew. In addition, vorozole treatment lowers aromatase activity in male and female preoptic area, and reduces plasma estradiol concentrations in females. Our findings confirm and extend results demonstrated in other species, conducted with the active enantiomer (R83842), or the racemic mixture (R76713, racemic vorozole). We also report that vorozole treatment affects the immunocytochemical distribution of aromatase immunoreactivity (AROM-ir) in musk shrew brain. The histological identification of neurons that contain this enzyme has been difficult in mammals. Several aromatase enzyme antisera have been developed and used in brain, and each gives a different pattern of immunoreactivity. Moreover, despite the fact that aromatase activity is very high in the bed nucleus of the stria terminalis, several amygdala nuclei, the preoptic area and hypothalamus, AROM-ir in these regions has been very limited. The distribution of AROM-ir in female musk shew brain tissues is modified by treatment with vorozole prior to sacrifice. Female musk shrew brains contain aromatase immunoreactive cell bodies, as reported previously, in the central amygdala, lateral septum and to a limited extent in the bed nucleus of the stria terminalis (BST). Brains of females treated with vorozole show additional immunoreactivity in the preoptic area, hypothalamus, and medial amygdala, and have a broad distribution of AROM-ir in several subdivisions of the BST. Several sexual dimorphisms are apparent in musk shrews brains after treatment with vorozole. We have quantified this sexual dimorphism in the medial preoptic area (MPO) by counting immunoreactive cells. In both the rostral and caudal portions of the MPO, female brains contain significantly fewer AROM-ir cell bodies than males. These data are in complete agreement with sex differences in biochemical analyses of aromatase activity in the MPO. At this time we do not know if these dimorphisms are the result of differences in circulating levels of steroids in males and females, and/or if the AROM-ir nuclei regulate sexually dimorphic behaviors.

Animals↗

PubMed1996

Endocrine correlates of partner preference behavior in rams.

We studied a unique group of rams that would not mate with estrous ewes during extensive testing for sexual behavior. The same rams courted males in preference to females in 30-min sexual preference tests and were classified as male-oriented (n = 6). We compared the following endocrine profiles: systemic steroid concentrations, the capacity of the testes to biosynthesize 17 alpha-hydroxyprogesterone and testosterone from 3H-progesterone in vitro, and the levels of brain aromatase activity (AA) in male-oriented rams vs. rams that were proven breeders and designated as female-oriented (n = 7). After the last behavioral test, sera were collected, and males in each experimental group were killed. Brains and testes were obtained for subsequent determinations of AA and measurements of steroidogenic enzyme activity. All dissections and subsequent assays were performed without knowledge of experimental group assignments. Serum concentration of testosterone (T), dihydrotestosterone (DHT), androstenedione, estrone (E1), and estradiol-17 beta (E2) were determined by RIA. AA was quantified by a 3H2O assay validated for neural tissue of the ram. We studied frontal, parietal and cingulate cortex, cerebellum, hippocampus, olfactory bulb, septum, amygdala, infundibulum-median eminence, and preoptic area (POA). Serum T, E1, and E2 concentrations of female-oriented subjects were significantly higher (p < 0.05) than those in male-oriented subjects (SEM: 1559 +/- 228, 46 +/- 2, and 15 +/- 3 pg/ml vs. 874 +/- 196, 40 +/- 2, and 8 +/- 1 pg/ml serum, respectively). DHT and androstenedione concentrations in the systemic circulation did not differ between groups. Likewise, biosynthesis of labeled T and 17 alpha-hydroxyprogesterone from 3H-progesterone by testicular homogenates in vitro was significantly higher (p < 0.05) in female-oriented than in male-oriented subjects (28.8 +/- 8.1 vs. 12.1 +/- 2.3 mumol.h-1.mg protein-1 for T and 416.9 +/- 100.8 vs. 186.3 +/- 30.7 mumol.h-1.mg protein-1 for 17 alpha-hydroxyprogesterone). The highest level of AA was found in the POA, which was significantly greater in female-oriented than in male-oriented rams (472 +/- 34 vs. 296 +/- 24 fmol 3H2O.h-1.mg protein-1, p < 0.05). AA in other brain areas did not differ between experimental groups. Our data suggest that the testes of the male-oriented ram have reduced capacity for T production. In other species, T controls in situ estrogen formation not only by providing substrate for aromatization but also by up-regulating P450arom mRNA in the POA. Because the POA is part of a neural circuitry that mediates male sexual behavior in many species, we hypothesize that the capacity for aromatization influences sexual orientation of these rams.

17-alpha-Hydroxyprogesterone↗

PubMed1996

Sex differences in androgen responsiveness in the rat brain: regional differences in the induction of aromatase activity.

The transformation of testosterone (T) to estrogens in brain tissue by cytochrome P-450 aromatase is required for the expression of sexual behaviors in adult male rats. Androgens regulate aromatase activity in the medial preoptic nucleus (MPN), as well as in a reciprocally connected group of forebrain nuclei involved in the regulation of male sexual behaviors. The levels of aromatase in these nuclei are generally greater in males than in females due to sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic. The current experiments were undertaken: (1) to characterize and compare the kinetic properties of aromatase in male and female rats and (2) to study sex differences in the dose-response relationship between the administered doses of T and the induction of aromatase in microdissected brain regions. Saturation analysis of aromatase activity in the MPN, bed nucleus of the stria terminalis (BNST), periventricular preoptic area (PVPOA), anterior hypothalamus (AH), and ventromedial hypothalamic nucleus (VMN) indicates that the greater aromatase activity observed in intact males reflects a sex difference in the maximal enzyme velocity, and not a sex difference in the apparent affinity of enzyme for substrate (Michaelis constant). The dose-response study of aromatase induction in the BNST, PVPOA, and VMN indicated a sex difference in aromatase activity over a range of circulating T levels varying from 0.3 to 35 ng/ml. No sex difference in inducible aromatase activity in AH was observed at any dose of T. The results of this study clearly demonstrate a sexually dimorphic effect of androgen action in the rat brain. Since T both regulates and is the substrate for aromatase in the brain, this sexual dimorphism is potentially an important limitation to the action of T in females and may relate to the enhanced expression of T-stimulated copulatory behavior in males compared to females.

Animals↗

PubMed1995

Subcellular localization and kinetic properties of aromatase activity in rat brain.

The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (approximately 10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

Amygdala↗

PubMed1994

Aromatase activity in developing guinea pig brain: ontogeny and effects of exogenous androgens.

The formation of estrogens from androgens by aromatase in the developing brain is an important step in the sexual differentiation of many species. We characterized aromatase activity (AA) in a high-speed pellet of brain tissue from fetal guinea pigs. The apparent substrate affinity (approximately 17 nM) was comparable to reported values in other species. Aromatase activity was highest in the amygdala (AMG) and preoptic area (POA), with lesser amounts in the septum (SEPT) and medial basal hypothalamus (MBH). Activity was low but measurable in parietal cortex (CTX). In the AMG, POA, SEPT, and MBH, AA was highest in early gestation (Days 35-40) and showed a steady decline through development. No sex difference in AA was apparent. We also determined the effects of administration of exogenous androgens to pregnant females on brain AA in the fetus. Testosterone propionate (5 mg/day on Days 30-39 followed by 1 mg/day on Days 40-50) caused a significant increase (p < 0.05) in AA found in the MBH and CTX. Administration of dihydrotestosterone propionate (2.5 mg/day on Days 30-39 followed by 1 mg/day on Days 40-50) significantly stimulated AA in SEPT, MBH, and CTX. These data demonstrate that the fetal guinea pig brain contains high levels of AA during the critical period of sexual differentiation. Treatment with high levels of exogenous androgens consistently induces AA in the MBH and CTX. These latter effects may be among the mechanisms through which exogenous androgens act on the developing brain.

Amygdala↗

PubMed1994

Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain.

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450AROM) is an important step in the mechanism of androgen action in the brain. In adult rats, P450AROM activity (AA) is regulated by androgens in the preoptic area and medial basal hypothalamus, but is constitutive in the amygdala. This study was undertaken to determine the distribution of P450AROM messenger RNA (mRNA) and AA in adult rat brain and examine the effects of steroid treatments on their concentrations in various brain regions. AA was determined by a sensitive assay that measures the production of 3H2O during the conversion of [1 beta-3H]androstenedione to estrone. P450AROM mRNA was measured by a ribonuclease protection assay using a RNA probe complementary to the 5'-coding region of rat P450AROM mRNA. The 32P-labeled P450AROM probe protected two mRNA fragments in brain tissues that expressed AA (preoptic area, medial basal hypothalamus, amygdala, and hippocampus). The larger protected RNA fragment was 430 nucleotides (nt) long and corresponded in size to the full-length protected complementary RNA, whereas the shorter protected RNA fragment was 300 nt long. Brain tissues that did not exhibit AA contained either the smaller protected RNA fragment (cingulate and parietal cortex) or no protected RNA (cerebellum). These results suggest that the 430-nt protected RNA fragment represents mRNA that encodes the functional P450AROM enzyme. In agreement with this conclusion, we found that immature rat ovaries that were stimulated with PMSG to synthesize estrogen contained only the 430-nt protected fragment. The levels of the 430-nt protected RNA fragment differed significantly between brain regions (amygdala > > preoptic area > medial basal hypothalamus > or = hippocampus) and were significantly correlated with AA (r = 0.994; P < 0.001). After castration, the concentrations of P450AROM mRNA and AA decreased significantly in the preoptic area and medial basal hypothalamus (P < 0.05), but not in the amygdala. Treatments with testosterone or dihydrotestosterone maintained P450AROM mRNA and AA at levels approximating those found in intact males. Although 17 beta-estradiol treatment increased AA in the preoptic area, it did not affect the P450AROM mRNA content. These results suggest that the increase in AA observed after exposure to androgens results from regulation of the transcription and/or stability of P450AROM mRNA. In contrast, estradiol appears to exert an effect on AA at the posttranscriptional level.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗

PubMed1993

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.

Amygdala↗

PubMed1993

Age-related deficits in brain estrogen receptors and sexual behavior of male rats.

Neural estrogen receptors (ER), serum testosterone (T), estradiol (E2) and luteinizing hormone (LH), and masculine sexual behavior were measured in young (5 months) and old (24 1/2 months) Fischer 344 male rats. We found that old intact males, which displayed significantly lower levels of sexual behavior, T, and LH than young intact males, also had lower levels of nuclear ER (ERn) in the amygdala (AMG). The age difference in ER binding did not appear to be a consequence of altered blood E2 levels because circulating E2 did not differ between the two age groups. Gonadectomy eliminated ejaculatory behavior and significantly reduced ERn in young males. When we administered exogenous T to gonadectomized males in doses that approximated levels found in young intact males, we found that sexual performance of old males was stimulated to precastration levels but not to levels found in young males. Moreover, such treatment failed to increase ERn in the AMG of old males to the levels measured in the AMG of young males. These results suggest that there is an association between the inability of T to increase ERn concentration in the AMG and the deficits in sexual performance that are characteristic of old males. Thus, the capacity of neural tissue to bind estrogen, presumably derived from circulating T, may be a limiting factor in the determination of androgen responsiveness in aging males.

Aging↗

PubMed1993

Selective activation of androgen receptors in the subcortical brain of male cynomolgus macaques by physiological hormone levels and its relationship to androgen-dependent aromatase activity.

Aromatase activity (AA) is androgen dependent and independent in subcortical regions of the nonhuman primate brain, but the correlation of androgen receptor (AR) content with AA has not been demonstrated. Thus, we castrated 10 adult male cynomolgus monkeys (Macaca fascicularis) and divided them into 2 groups. One group (n = 6) received empty Silastic capsules, whereas the second group (n = 4) received Silastic capsules filled with testosterone (T). Animals were killed after 3 weeks. Microsomal AA and cytosolic and nuclear AR were determined in specific brain regions dissected from frozen sections. Sera from T-treated subjects contained T, dihydrotestosterone, and LH levels that were not significantly different from the precastration amounts (P < 0.05). Cytosolic AR concentrations declined after T treatment in 12 of 20 brain areas studied (P < 0.05). Nuclear AR levels, on the other hand, were significantly elevated after T treatment (activated) only in the ventral medial nucleus (VMN) and infundibular nucleus/median eminence (P < 0.05). AA distribution was significantly different (P < 0.05) among 20 brain nuclei and subregions. The highest activities were found in the bed nucleus of the stria terminalis, the medial preoptic area, the medial and cortical amygdala, and the VMN. Lesser activities were found in other brain regions. Physiological concentrations of T increased AA only in the VMN and infundibular nucleus-median eminence (P < 0.05). These data suggest that physiological levels of androgens are effective in regulating AA only in those brain areas in which AR are activated.

Androgens↗

PubMed1993

Differential effects of aromatase inhibition on luteinizing hormone secretion in intact and castrated male cynomolgus macaques.

To understand the role of central aromatization in feedback regulation of LH in nonhuman primates, we treated adult male cynomolgus monkeys with the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD). We measured LH, testosterone (T), and ATD in systemic sera of blood samples drawn on a diurnal schedule (0900 and 2100 h). Each animal was bled for 4 pretreatment days from a femoral catheter after which they were divided into the following treatment groups: castrated (Cx), n = 2; Cx + T, n = 6; Cx + T + ATD, n = 6; Cx + ATD, n = 3; and sham operated + ATD, n = 3. Silastic capsules or packets containing T or ATD, respectively, were placed sc between the scapulae at the time of Cx or sham treatment. In T-treated animals, T (20 micrograms/kg body weight) dissolved in propylene glycol was injected im at 2100 h to mimic the diurnal rise of T observed in nonhuman primates. Animals were bled for 2 weeks after which they were killed, and selected brain areas were analyzed for aromatase activity and cytosolic and nuclear androgen receptors. Animals treated with ATD had significantly reduced levels of aromatase activity in selected regions of the hypothalamus, preoptic area, and the amygdala (P < 0.05). Even though ATD inhibited brain aromatase activity, it did not prevent the negative feedback actions of T on LH secretion after Cx. In addition, ATD by itself inhibited LH secretion after Cx and activated brain androgen receptors. These latter effects of ATD seemed to have been mediated through a metabolite. In sham-operated intact males, ATD produced variable surges of LH that were accompanied by elevations of T in the systemic circulation. These differential effects of ATD in intact vs. castrated animals demonstrate the importance of selecting the proper model system to study LH control mechanisms. In the intact animal, aromatization seems to play a role in regulating LH secretion, but the postcastration rise of LH seems to be regulated differently.

Androstatrienes↗