Androgen receptor expression in the developing male and female rat visual and prefrontal cortex.

Gonadal steroid hormones are known to influence the development of the cerebral cortex of mammals. Steroid hormone action involves hormone binding to cytoplasmic or nuclear receptors, followed by DNA binding and gene transcription. The goals of the present study were twofold: to determine whether androgen receptors are present during development in two known androgen sensitive regions of the rat cerebral cortex, the primary visual cortex (Oc1) and the anterior cingulate/frontal cortex (Cg1/Fr2); and to determine whether androgen receptor (AR) expression in these regions differs between developing males and females. We used immunocytochemistry to detect AR protein on postnatal days 0, 4, and 10, and in situ hybridization to detect AR mRNA on postnatal day 10 in male and female rats. The level of AR expression was specific to the cortical region, with higher AR immunoreactive cell density and more AR mRNA in Oc1 than in Cg1/Fr2. AR immunoreactive cell density increased with age in both regions. Finally, on postnatal day 10, males had a higher AR immunoreactive cell density and more AR mRNA in Oc1 than did females. Thus, the presence of ARs may allow androgens to directly influence the development the cerebral cortex.

Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem.

Dioxin exposure alters a variety of neural functions, most likely through activation of the arylhydrocarbon receptor (AhR) pathway. Many of the adverse effects, including disruption of circadian changes in hormone release and depressed appetite, seem to be mediated by hypothalamic and/or brainstem neurons. However, it is unclear whether these effects are direct or indirect, because there have been no comprehensive studies mapping the expression of components of the AhR pathway in the brain. Therefore, we used a sensitive in situ hybridization histochemical (ISHH) method to map the neural expression of AhR mRNA, as well as those of the mRNAs encoding the AhR dimerization partners, arylhydrocarbon receptor nuclear translocator (ARNT) and ARNT2. We found that AhR, ARNT, and ARNT2 mRNAs were widely distributed throughout the brain and brainstem. There was no neuroanatomic evidence that AhR is preferentially colocalized with ARNT or ARNT2. However, ARNT2, unlike ARNT expression, was relatively high in most regions. The most noteworthy regions in which we found AhR, ARNT, and ARNT2 mRNA were several hypothalamic and brainstem regions involved in the regulation of appetite and circadian rhythms, functions that are disrupted by dioxin exposure. These regions included the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), nucleus of the solitary tract (NTS), and the dorsal and median raphe nuclei. This neuroanatomic information provides important clues as to the sites and mechanisms underlying the previously unexplained effects of dioxins in the central nervous system.

Estrogen, but not androgens, regulates androgen receptor messenger ribonucleic acid expression in the developing male rat forebrain.

Testosterone is the principal gonadal hormone responsible for the masculinization of the rat nervous system. Sex differences in both the ligand and receptor availability may play a role in the process of sexual differentiation. In some brain regions, males express more androgen receptor (AR) messenger RNA (mRNA) than females by postnatal day (PND) 10. Gonadectomy on the day of birth (PND-0) eliminated the sex differences in AR mRNA expression at PND-10, and exogenous testosterone replacement restored this sex difference. Because testosterone can be converted to both androgenic and estrogenic metabolites in the brain, the present experiments were performed to determine whether androgenic or estrogenic metabolites of testosterone are responsible for region-specific regulation of AR mRNA content in the developing rat forebrain. We used a 35S-labeled riboprobe and in situ hybridization to assess relative steady-state levels of AR mRNA in animals killed on PND-10. In the principal portion of the bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), males gonadectomized on PND-0 and treated daily with dihydrotestosterone propionate (DHTP), a nonaromatizable androgen, had low levels of AR mRNA that were not significantly different from AR mRNA levels in intact females. In contrast, males gonadectomized on PND-0 and treated daily with diethylstilbestrol (DES), a synthetic estrogen, maintained high, male-typical levels of AR mRNA in the BSTpr and the MPO. AR mRNA content in the VMH was not sexually differentiated in PND-10 rats and was unaffected by gonadectomy or hormone replacement. To further assess whether AR mRNA was autologously regulated, neonatal male rats were treated with the androgen receptor antagonist, flutamide. Flutamide at a dose of either 40 microg/day or 300 microg/day had no effect on AR mRNA expression in any area examined. Thus, AR mRNA is up-regulated by estrogen but is not regulated by androgen during the early postnatal period.

Regulation of androgen receptor messenger ribonucleic acid expression in the developing rat forebrain.

By postnatal day 10 (PND-10), males express more androgen receptor (AR) messenger RNA (mRNA) than females in the principal portion of the bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), but not in the ventromedial hypothalamus. The development of these region-specific sex differences in AR mRNA expression may be critical for the organization of male-typical neural circuitry and may represent the onset of sex differences in the sensitivity of the rat brain to the actions of androgens. In this study, we used a 35S-labeled riboprobe and in situ hybridization to address whether postnatal testosterone exposure is important for the up-regulation of AR mRNA content in the developing rat forebrain. In the BSTpr and the MPO of PND-10 rats, males gonadectomized on PND-0 or PND-5 had lower levels of AR mRNA compared with intact or sham-operated control males. Daily replacement of testosterone to animals gonadectomized on PND-0 maintained AR mRNA content in the BSTpr and the MPO at levels equal to those in intact males. In contrast, there was no effect of gonadectomy or testosterone replacement on AR mRNA expression in the ventromedial hypothalamus. Thus, the postnatal hormonal environment may permit the development of region-specific sex differences in AR mRNA. Significant alterations in AR mRNA expression in the BSTpr and MPO in PND-10 male rats were induced by gonadectomy as late as PND-8. Males gonadectomized on PND-8 had levels of AR mRNA significantly lower than those in intact males, but significantly higher than those in intact females. Further, when animals were gonadectomized on PND-0 and given testosterone on PND-8 and PND-9, levels of AR mRNA were also intermediate between those found in intact males and intact females. The exact time course for transcriptional regulation of AR mRNA in the developing rat brain is unknown. However, others have shown significant regulation of AR mRNA within hours of hormone treatment, so that 2 days of hormone withdrawal or replacement are probably sufficient to achieve new steady state levels of message. Moreover, sexually dimorphic neuronal loss has been documented to peak in hypothalamic cell groups during the first postnatal week. Thus, it is likely that changes in the number of AR mRNA-expressing cells as well as the amount of AR mRNA expression per cell are responsible for the development of male-typical AR mRNA content.

Ontogeny of region-specific sex differences in androgen receptor messenger ribonucleic acid expression in the rat forebrain.

Testosterone and its metabolites are the principal gonadal hormones responsible for sexual differentiation of the brain. However, the relative roles of the androgen receptor (AR) vs. the estrogen receptor in specific aspects of this process remain unclear due to the intracellular metabolism of testosterone to active androgenic and estrogenic compounds. In this study, we used an 35S-labeled riboprobe and in situ hybridization to analyze steady state, relative levels of AR messenger RNA (mRNA) expression in the developing bed nucleus of the stria terminalis, medial preoptic area, and lateral septum, as well as the ventromedial and arcuate nuclei of the hypothalamus. Each area was examined on embryonic day 20 and postnatal days 0, 4, 10, and 20 to produce a developmental profile of AR mRNA expression. AR mRNA hybridization was present on embryonic day 20 in all areas analyzed. In addition, AR mRNA expression increased throughout the perinatal period in all areas examined in both males and females. However, between postnatal days 4 and 10, sharp increases in AR mRNA expression in the principal portion of the bed nucleus of the stria terminalis and the medial preoptic area occurred in the male that were not paralleled in the female. Subsequently, males exhibited higher levels of AR mRNA than females in these areas by postnatal day 10. There was no sex difference in AR mRNA content in the lateral septum, ventromedial nucleus, or arcuate nucleus at any age. These results suggest that sex differences in AR mRNA expression during development may lead to an early sex difference in sensitivity to the potential masculinizing effects of androgen.

Estrogen receptor mRNA levels in the preoptic area of neonatal rats are responsive to hormone manipulation.

Testosterone, after conversion to estrogen, masculinizes the developing preoptic area (POA) of rats, via binding to intracellular estrogen receptors located within the POA. Our previous studies have shown what seems to be a paradox, in that the levels of estrogen receptor mRNA are lower in males than in females. In the present study, we examined the effects of hormone manipulations on estrogen receptor (ER) mRNA levels in the preoptic area of neonatal male and female rats to test the hypothesis that gonadal steroid hormones regulate ER mRNA during the perinatal period. The relative amount of steady state ER mRNA was assessed in the preoptic area of postnatal day 4 animals using in situ hybridization and film autoradiography. Hybridization density was approximately 2-fold higher in females compared with hybridization density in males. Depletion of testosterone by bilateral removal of the testes on the day of birth increased the level of ER mRNA in males to that observed in females. Treatment of females with the synthetic estrogen, diethylstilbestrol (1 microgram per day, in pellet form), reduced ER mRNA levels to a level comparable to that in intact males. The non-aromatizable androgen, dihydrotestosterone (50 micrograms per day, in pellet form), had no effect on ER mRNA in females. These results suggest that estrogen, derived from the local aromatization of circulating testosterone, down-regulates ER mRNA in the neonatal male preoptic area. Down-regulation of ER mRNA may be an important estrogen-regulated event in the process of sexual differentiation of the preoptic area.