Effects of Cyclic AMP and Andre‐gens on in vitro Brain Aromatase Enzyme Activity During Prenatal Development in the Rat

Abstract

In the rat, there is a marked but transient increase in hypothalamic aromatase activity during the last week of fetal life. The present study was undertaken to gain insight into the regulation of this developmental pattern. Hypothalamic fragments comprising the medial basal hypothalamus and the suprachiasmatic region (henceforth referred to as preoptic area) were explanted and cultured in serum-free medium for 2 to 5 days. Aromatase activity was measured by the formation of (3) H(2) O, utilizing either [1ß-(3) H]androstene-dione or [1ß-(3) H]testosterone as substrate. Maximal rates of activity were obtained at a saturating concentration of 0.3 μM [1ß-(3) H]testosterone. Confirmation of the identity of the [(3) H]estradiol formed was demonstrated by recrystallization of the derivatized estradiol to constant specific activity following incubation with [1,2,6,7-(3) H]testosterone. In agreement with previous reports, in vivo hypothalamic aromatase activity was negligible before gestational day (GD) 16, increased strikingly by GD19 (>5.0 pmol/h/mg protein) and decreased, thereafter, to low levels at GD22 (∼1.0 pmol/h/mg protein). Medial basal hypothalamus-preoptic area fragments explanted before GD17 failed to develop aromatase activity in vitro. If the tissue was explanted on GD17 or 18 (i.e. when the in vivo rate of activity was increasing), the enzyme activity did not continue to increase, but it was rather maintained for 2 days before decreasing in a manner that closely mimicked the decline observed in vivo. A similar, butimmediate decline was observed when the tissue was explanted on GD19 (i.e. at the time when theactivity peaks in vivo). Exposure of explants to either growth factors (insulin-like growth factor II, epidermal growth factor, and basic or acidic fibroblast growth factor), or steroids (estradiol-17ß, progesterone, testosterone, dihydrotestosterone and corticosterone) failed to either increase aromatase activity before the peak at GD19 or ameliorate its perinatal decline. Increase of Ca(2+) fluxes with the ionophore A23187 or activation of the cyclic AMP, cyclic GMP, or protein kinase C pathways were similarly ineffective, as was angiotensin II, a recently proposed stimulator of neural aromatase. In contrast, aromatase activity was suppressed 2- to 4-fold by activation of the cyclic AMP pathway (with either forskolin or 8-bromo-cyclic AMP) or by the androgens, testosterone and dihydrotestosterone. These results suggest that: 1) the appearance of aromatase activity in the rat hypothalamus before GD17 requires the unfolding of extrahypothalamic events, 2) the increase in aromatase activity that occurs before GD19 also requires extrahypothalamic inputs and does not involve any of the known intracellular signal transduction pathways, and 3) the decline in activity observed after GD19 is regulated within the hypothalamus, and appears to be determined, at least in part, by the activation of cyclic AMP formation. A potential role for androgens is discussed.

Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish

“…For example, up-regulation of AroB transcripts following androgen treatment has been shown in mammals, ricefield eel, and in teleosts such as zebrafish and channel catfish; direct AR regulation of AroB has been demonstrated in ricefield eel, but in teleosts increased AroB expression is attributed to signaling by estrogenic metabolites and not to ARs directly (41,43,(54)(55)(56). In rodents (mice and rats), however, results of AR-mediated changes in AroB expression are mixed, with reports that androgens increase, decrease, or have no effect on AroB transcription (57)(58)(59)(60). Interestingly, AREs have been identified in the promoter of AroB (cyp19a1b) in teleosts and humans, suggesting that AR may modulate AroB directly; however, AREs have not been identified in murine animals, perhaps suggesting that there is an indirect mechanism of AroB modulation in these species (43,44).…”

In vitro Increase of Aromatase mRNA in Diencephalic Neurons

“…3B, C, 5C). Lephart et al [7] have also observed the decline in aromatase activity and reported that the elevation of cAMP formation was at least partly responsible for the decrease. In the present study, the NF-L mRNA level was still increasing when the aromatase mRNA had begun to decrease in culture ( fig.…”

“…Only two cases of the increase of aromatase activity in brain cells during the cul ture period have been reported: that in adult turtle in which the brain aromatase level should be constant rather than increasing time-dependently [26] and that in the murine hypothalamic cells [27], Recently. Lephart et al [7] studied aromatase activity in cultured tissue fragments from the medial basal hypothalamus and preoptic area of rats at various perinatal developmental stages. They did not, however, observe any increase of the enzyme activity in rat brain fragments cultured in vitro, whereas both aro matase activity and its mRNA in rat brain increased strik ingly with a transient peak at E19-E20 in vivo [7,9], The differences in species, developmental stages at which cells were prepared, and culture conditions may account for these discrepancies.…”

“…Lephart et al [7] studied aromatase activity in cultured tissue fragments from the medial basal hypothalamus and preoptic area of rats at various perinatal developmental stages. They did not, however, observe any increase of the enzyme activity in rat brain fragments cultured in vitro, whereas both aro matase activity and its mRNA in rat brain increased strik ingly with a transient peak at E19-E20 in vivo [7,9], The differences in species, developmental stages at which cells were prepared, and culture conditions may account for these discrepancies.…”

“…Furthermore, estrogens formed in situ are thought to be decisive for these effects [2,3]. Aromatase (estrogen synthetase, EC 1.14.14.1), which catalyzes the conversion of androgens to estrogens, is Aromatase activity has been found in the brains of many species of vertebrates [4,5], Aromatase activity [6][7][8][9] and the steady state level of aromatase mRNA [9,10] in rodent brains increase transiently during perinatal devel opment with a peak at the critical period for neonatal imprinting of sex-related expression [11], However, very little is known about the factors that control the increase of brain aromatase expression during prenatal development.…”