Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain

Abstract

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Thus, brain 3beta-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3beta-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3beta-HSD, we asked whether 17beta-estradiol (E(2)) regulates DHEA metabolism in adult zebra finch (Taeniopygia guttata) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [(3)H]DHEA and radioinert E(2)in vitro. Within only 10 min, E(2) significantly reduced 3beta-HSD activity in both male and female brain. Interestingly, the rapid effects of E(2) were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.

Fig. 1

Simplified diagram of sex steroid synthesis. Steroids: PREG, pregnenolone; PROG, progesterone. Enzymes: CYP11A1, cytochrome P450 side chain cleavage; CYP17, cytochrome P450 17α-hydroxylase/C17, 20 lyase. The enzyme 3β-HSD metabolizes DHEA into AE. AE can then be converted to other steroids: 5β-A, 5β-androstanedione; 5α-A, 5α-androstanedione; E₁, estrone; T, testosterone.

Fig. 2

Timecourse of DHEA metabolism by 3β-HSD in adult male zebra finch brain. Incubations were carried out at 41°C with 200 nmol/L [³H]DHEA and 1 mmol/L NAD+, and products were separated using TLC. (a) DHEA metabolism in brain homogenate and supernatant from 15 to 180 min. Open symbols represent product formation in supernatants, and closed symbols represent product formation in homogenates. n = 3 replicates per timepoint. (b) DHEA metabolism in brain supernatants from 2.5 to 15 min. n = 2 replicates per timepoint.

Fig. 3

Effect of trilostane, a specific 3β-HSD inhibitor, on 3β-HSD activity in adult male zebra finch brain. [³H]DHEA metabolism to [³H]AE was determined by (a) HPLC and (b) TLC analyses. n = 4 replicates per group.

Fig. 4

Effect of in vitro E₂ on DHEA metabolism by 3β-HSD in adult male zebra finch brain, as measured by TLC. E₂ concentrations ranged from 0 to 2000 nmol/L. n = 3 replicates per group.

Fig. 5

Representative HPLC chromatograph illustrating the peaks and retention times of [³H]AE, [³H]UNK (an unknown steroid), and [³H]DHEA. [³H]UNK peak area was not affected by trilostane or estradiol.

Fig. 6

HPLC analysis of the rapid effect of E₂ on 3β-HSD activity in adult male and female zebra finch brain. 3β-HSD activity in the presence of 0, 4, or 1000 nmol/L E₂ for 10 min was determined using HPLC for steroid separation and product quantification. n = 6 males and 6 females. **p < 0.01.

Fig. 7

TLC analysis of the rapid effect of E₂ on 3β-HSD activity in adult male and female zebra finch brain. 3β-HSD activity in the presence of 0, 4, or 1000 nmol/L E₂ for 10 min was determined using TLC for steroid separation. n = 6 males and 6 females. (a) Effect of E₂ on total 3β-HSD metabolites ([³H]AE and [³H]5β-A). (b) Effect of E₂ on only [³H]5β-A levels. **p < 0.01, ***p < 0.001.

Fig. 8

Effect of 1000 nmol/L E₂ on 3β-HSD activity as determined by HPLC and TLC analyses. All data are expressed relative to within-subject baseline (0 nmol/L E₂). TLC analysis revealed a significant sex difference in inhibition of 3β-HSD activity by E₂. n = 6 males and 6 females. *p < 0.05.

Fig. 9

[³H]AE and [³H]5β-A expressed as a percentage of total 3β-HSD metabolites, as determined by TLC. n = 6 males and 6 females. *p < 0.05, **p < 0.01.