Brain Steroid Metabolism
Regulation of Aromatase Gene Expression in the Adult Rat Brain

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Abstract

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.

Introduction

Testosterone (T) is metabolized to estradiol within the CNS through the activity of a specific cytochrome P450 enzyme named aromatase [24]. In rats, central aromatization is required for sexual differentiation of the developing brain 22, 23and the display of male sexual behavior 4, 5. In primates, part of the feedback exerted by T on gonadotropin secretion is mediated through aromatization [7].

In vitro assays have demonstrated the presence of aromatase activity in many regions of the male rat brain 11, 35. The most prominent nuclei are the bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventromedial nucleus of the hypothalamus (VMN), and the medial and cortical amygdala (MA and CA). These nuclei are reciprocally connected and are important components of a sexually dimorphic neural circuitry that controls male sexual behavior 34, 36, 37. Consistent with its pattern of distribution, inhibition of aromatase activity within the preoptic area suppresses male sexual performance [5].

Androgens regulate aromatase activity in some, but not all areas of the brain. The activity in discrete nuclei of the preoptic area (POA) and medial basal hypothalamus (MBH) is decreased by castration and restored to intact levels by treatment with T or dihydrotestosterone (DHT) 28, 30. The stimulation of enzyme activity by androgen is blocked by concomitant antiandrogen administration [30], suggesting that T acts through a specific androgen–receptor mechanism. Further support for this concept is derived from studies showing that aromatase activity is significantly lower in males that are genetically androgen receptor deficient (i.e., testicular feminized) than in their normal litter mates [32]. In contrast, aromatase in the amygdala is not consistently affected by castration and T treatment. Microdissection studies suggest that androgen regulation may exist within specific cell populations of the MA but not CA [27]. The mechanisms accounting for the region-specific regulation of aromatase activity in the brain remain to be determined.

There are substantial sex differences in aromatase activity in many brain areas [27]. In the BNST, MPN, and VMN, aromatase activity is approximately two to four times greater in males than in females. At least part of the gender difference can be explained by the fact that males are exposed to greater quantities of circulating androgens than females. The sex difference is abolished when males are castrated and reinstated when castrated males are treated with T. Interestingly, the mechanism of aromatase induction appears to be sexually dimorphic [25]. Treatment with physiological doses of T stimulates aromatase activity in BNST, MPN, and VMN to a greater extent in males than in females. The sex difference in activity is characterized by a difference in the Vmax, but not the Km, signifying that it is due to differences in enzyme concentrations [26]. Dose–response studies indicate that the sex difference in aromatase induction is apparent over a range of circulating T concentrations and constitutes a gender difference in T efficacy, but not potency [29]. Thus, the greater capacity for aromatization exhibited by males appears to be a sexually differentiated feature of the brain that could account for gender differences in the responsiveness of adults to androgens.

The recent cloning of the aromatase gene has greatly advanced our understanding of the regulatory, structural, and functional features of this enzyme [38], Brain aromatase mRNA was first detected in the perinatal rat brain by Lephart et al., who identified a single 2.7 KB transcript [18]that is similar in size to the largest functional aromatase mRNA species previously observed in rat ovarian tissue [19]. The levels of brain aromatase have been primarily examined during perinatal development 13, 16, 17, 20, 42. This is most likely because of the relative abundance of aromatase activity in brain during perinatal life and the important role it plays in the sexual differentiation of the brain. In contrast, less is known about aromatase mRNA expression in the adult nervous system 1, 26, 40. A major obstacle to its study in adults is the extremely low levels of expression found in the mature brain. For this reason, we employed a sensitive sequence-specific ribonuclease protection assay to measure aromatase mRNA in the adult rat brain. This report summarizes recent work from our laboratory in which we found that adult brain regions contain differing levels of functional aromatase mRNA, as well as a unique aromatase mRNA transcript of unknown function. Moreover, our results demonstrate that androgens regulate aromatase activity by regulating the transcription or stability of aromatase mRNA. Finally, we found that sex differences in aromatase regulation are exerted pretranslationally, demonstrating that gender differences in androgen responsiveness affect gene expression in the adult rat brain.

Section snippets

Animals and Tissue Dissections

Adult male and female Sprague–Dawley rats (60–70 days of age) were used in these studies. The care and experimental use of the rats were conducted in accordance with guidelines specified in the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee of the Oregon Health Science University. Surgeries were performed on rats anesthetized with ketamine (55 mg/kg) and xylazine (5 mg/kg). When steroids were

Distribution of Aromatase mRNA

The aromatase cRNA probe used in our studies protected two RNA fragments in the adult rat brain; a 430 nt full-length fragment and a shorter 300 nt fragment (Fig. 1). Brain tissues that exhibit aromatase activity contained both protected fragments (i.e., POA, MBH, amygdala, and hippocampus. Brain areas that do not exhibit measurable aromatase activity contained either the smaller fragment (i.e., cingulate and parietal cortex), or no protected RNA (cerebellum). The ovary contained only the 430

Discussion

These experiments demonstrate the presence of an alternate aromatase mRNA transcript in the adult rat brain. The aromatase cRNA probe that we used protected a mRNA fragment (430 nt) corresponding to both a full-length transcribed sense RNA and a truncated fragment of approximately 300 nt. The distribution of the full-length mRNA fragment was significantly correlated with enzymatic activity and is believed to represent the mRNA encoding functional enzyme. In contrast, the truncated mRNA fragment

Acknowledgements

Portions of this work supported by NSF Grant IBN 94-21759 (CER) and NIH Grants HD-18196 (C. E. R.), HD-23293 (J. A. R.) and P30-HD-18185. We wish to recognized the expert technical assistance of Henry Stadelman, Emile Jorgensen, and Scott Klosterman. We also wish to thank Drs. Edwin Lephart, Michael McPhaul, and Serjio Ojeda for providing the aromatase cDNA probes.

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