The organizational and aromatization hypotheses apply to rapid, nonclassical hormone action: neonatal masculinization eliminates rapid estradiol action in female hippocampal neurons

Abstract

Early exposure to the steroid sex hormone testosterone and its estrogen metabolite estradiol masculinize neural tissue during a developmental critical period. Many aspects of neuron anatomy and physiology are permanently altered, including later sensitivity to estradiol. Although it is well established that early hormone exposure alters neuronal responsiveness regarding classical estradiol actions (i.e. acting via nuclear estrogen receptors), it has not yet been determined whether it also alters neuronal processing of nonclassical estrogen receptor signaling, including the actions of membrane-associated estrogen receptors. Hence, we tested whether membrane estrogen receptor regulation of cAMP response element binding protein (CREB) phosphorylation observed in female (but not male) hippocampal pyramidal neurons is due to the lack of androgen and/or estrogen exposure in females during this critical period. Female rat neonates on postnatal d 0 and 1 were systemically injected with one of four compounds: vehicle, testosterone, the nonaromatizable androgen dihydrotestosterone, or estradiol. On postnatal d 2, primary hippocampal neuron cultures were generated from these animals. After 8-9 d in culture, we assessed whether estradiol affected CREB phosphorylation. Neurons from female neonates exposed to testosterone lacked estradiol signaling to CREB. In contrast, dihydrotestosterone injections of female neonates did not disrupt estradiol regulation of CREB. Estradiol injections of female neonates, however, eliminated estradiol signaling to CREB. These findings indicate that testosterone aromatization to estradiol leads to a masculinization/defeminization process whereby hippocampal neurons fail to exhibit rapid estradiol signaling to CREB. Broadly, these findings extend the organizational and aromatization hypotheses to rapid, nonclassical hormone action.

Fig. 1.

Rapid, nonclassical estradiol action on CREB phosphorylation is sex specific and is regulated by early hormone exposure. A, Example confocal images of cultured hippocampal neurons immunolabeled with MAP2 (green) and pCREB (red). Exposure to estradiol (E) rapidly increased CREB phosphorylation and decreased 20 mm K⁺-induced CREB phosphorylation in female neurons. Treatments were as follows: top left, vehicle; top right, estradiol; bottom left, 20 mm K⁺; bottom right, estradiol and 20 mm K⁺. Scale bar, 25 μm. B, Quantification of rapid estradiol modulation of CREB phosphorylation in female neurons. C, Estradiol exposure has no effect in male neurons. D, Neonatal exposure to testosterone (T) eliminates later responsiveness to rapid estradiol action in female neurons. E, Neonatal exposure to testosterone's nonaromatizable, androgenic metabolite dihydrotestosterone (DHT) does not eliminate later responsiveness to rapid estradiol action in female neurons. F, Neonatal exposure to testosterone's estrogenic metabolite estradiol eliminates later responsiveness to rapid estradiol action in female neurons. Letters within each bar indicate statistically significantly different groups; complete statistical information is in Results.

Fig. 2.

Neonatal exposure to testosterone (T) does not affect mGluR signaling. A, Neonatal exposure to oil does not affect group I mGluR signaling induced by the agonist DHPG or rapid estradiol (E) action in female neurons; B, neonatal exposure to testosterone does not eliminate group I mGluR signaling but does eliminate rapid estradiol action in female neurons; C, neonatal exposure to oil does not eliminate group II mGluR signaling induced by the agonist APDC (AP) in female neurons; D, neonatal exposure to testosterone does not affect group II mGluR signaling but does eliminate rapid estradiol action on pCREB intensity induced by 20 mm K⁺. Letters within each bar indicate statistically significantly different groups; complete statistical information is in Results.