Jerome V.A. Choate

Immunocytochemical localization of androgen receptors in brains of developing and adult male rhesus monkeys.

We localized immunoreactive androgen receptors in the central nervous system (CNS) of fetal and adult male rhesus macaques by immunocytochemisty using an affinity-purified polyclonal antibody to the first 21 amino acids of the human androgen receptor (AR). This antibody caused a shift in the mobility of AR-bound 3H-DHT on a sucrose gradient and recognized a protein of approx 116 kDa on Western blot. Other criteria for specificity are presented. We localized AR in the diencephalon of male rhesus monkey fetuses. Immunoreactive neurons were found in the medial hypothalamic area and the ventromedial nucleus of the hypothalamus on days 47, 61, and 124 of gestation. At 124 d of gestation, AR immunoreactivity was also found in the arcuate nucleus. AR immunostaining was not found in other diencephalic structures in fetal life, including the preoptic area. In the adult monkey, neurons in ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus; cortical, medial, and accessory basal nuclei of the amygdala; and regions of the hippocampus and the anterior pituitary gland contained immunoreactive AR. These data indicate that AR is found in specific areas of the CNS early in fetal development, but they also appear in other brain areas as the fetus grows. At 124 d of gestation (term, 167 d), the hypothalamic location of immunoreactive AR is similar to the adult.

Androgen receptor immunoreactivity in intact and castrate guinea pig using antipeptide antibodies

We developed and used antibodies to an androgen receptor (AR)-specific synthetic peptide (amino acids 201-222 of the human androgen receptor) to localize AR in the brain and peripheral tissues of intact and castrate adult male guinea pigs. Immunoreactivity was localized to the nucleus of epithelial and stromal cells in the prostate of intact animals. Immunostaining was abolished in the prostate 4 days following castration. Androgen receptor immunoreactivity was found in brain nuclei known to be involved in reproductive and other androgen-dependent behaviors, including the preoptic area, medial basal hypothalamus, and anterior pituitary gland. Castration had no effect on the distribution or intensity of AR immunostaining in the brain. These data indicate a differential regulation of AR in the brain compared to peripheral tissues.

Effects of androgen on brain and pituitary androgen receptors and LH secretion of male guinea pigs

To study the tissue-specific control of androgen receptors by circulating androgens, guinea pigs were castrated or castrated and treated with crystalline testosterone propionate. Levels of serum androgens and LH were measured and brain and peripheral tissues were collected for determination of androgen receptor levels under differing androgen states. We found that circulating androgen levels changed rapidly following castration or treatment with exogenous androgen. LH secretion was coupled to circulating androgens; high levels of androgen quickly suppressed LH secretion, whereas LH levels did not rise significantly above intact levels until 7 days following castration. Androgen receptor levels were effected by circulating androgens. Cytosolic androgen receptors (ARc) increased significantly in the preoptic area (POA), septum, anterior pituitary, prostate and seminal vesicle following castration, whereas nuclear androgen receptors (ARn) decreased significantly in the POA, septum, medial basal hypothalamus (MBH), amygdala, parietal cortex, pituitary, prostate and seminal vesicle. Exogenous androgens, which increased serum steroid levels significantly above that in intact animals, decreased ARc below control levels in MBH, amygdala, pituitary and seminal vesicle. High circulating androgens increased ARn above intact levels in the MBH, pituitary and prostate. It thus appears that circulating androgens regulate LH secretion and have profound, but tissue-specific effects on androgen receptors in the guinea pig.

Paradoxical effect of an aromatase inhibitor, CGS 20267, on aromatase activity in guinea pig brain

To determine the effect of in vivo treatment of guinea pigs with a non-steroidal aromatase inhibitor (CGS 20267; letrozole), we treated subjects with subcutaneous Silastic implants containing crystalline letrozole. We studied four treatment groups: intact, intact letrozole-treated, castrate and castrate letrozole-treated. After treatment for 1 week, brain tissues (preoptic area, septum, medial basal hypothalamus, amygdala and parietal cortex) were removed, and microsomal aromatase activity (AA) was determined by an in vitro 3H2O assay using 1beta-3H-androstenedione as substrate. Kinetic experiments were performed to determine the competitive nature of letrozole and an approximate Ki was calculated. Letrozole appears to be a reversible, competitive inhibitor of aromatase activity with an apparent Ki of 1.2 nM. Aromatase activity in intact letrozole-treated animals was elevated compared to untreated controls in all brain areas tested (P< 0.05). Letrozole also stimulated AA in the brains of letrozole-treated castrated guinea pigs compared to untreated castrated animals (P< 0.05). These data indicate that letrozole administered in vivo causes an increase in AA. Possible mechanisms include an autoregulatory mechanism which is interrupted by enzyme inhibition, or an effect of the inhibitor on turnover rates of P450 aromatase.

Effects of Exogenous Androgen on Brain Androgen Receptors of the Fetal Rhesus Monkey

Testosterone secreted by the fetal testes masculinizes and defeminizes the nonhuman primate brain during a defined prenatal critical period. We previously demonstrated the presence of high-affinity, specific androgen receptors (AR) in the developing rhesus monkey brain, but did not present data concerning their capacity for activation. To achieve this end, we analyzed the AR content in brains from intact and gonadectomized rhesus monkey fetuses at approximately 125 days of gestation, 2 h after injection of either 500 micrograms dihydrotestosterone (DHT) or vehicle directly into the fetus. After treatment, plasma DHT concentrations increased five-fold in the fetal circulation. In gonad-intact fetuses, cytosolic AR decreased in preoptic area, medial basal hypothalamus, and septum following DHT treatment. No significant effect of DHT treatment on nuclear AR was seen. In contrast, the increased level of DHT in the maternal circulation decreased cytosolic AR and increased nuclear AR of the maternal myometrium. In gonadectomized fetuses, DHT treatment decreased cytosolic AR as it did in the intact group. In contrast, a significant increase in nuclear AR was seen in preoptic area, medial basal hypothalamus, and tegmentum of these fetuses. Thus AR in fetal rhesus brain can be activated by DHT when the gonads are removed, but not in the intact fetuses. These data suggest that AR in the developing nervous system of rhesus macaques can be activated by exogenous androgen and hence are probably functional.