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.

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.

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.