Androgens influence sexual differentiation of embryonic mouse hypothalamic aromatase neurons in vitro

Estrogen formed perinatally in the brain from testicular androgen by aromatase is involved in the irreversible determination of male brain development. Perinatal sex differences in aromatase activity have been observed in the hypothalamus. Testosterone (T) is a major modulator for aromatase in the adult rat hypothalamus. However, it is not known whether circulating T influences aromatase neurons during fetal brain development. To study the influence of androgen exposure on embryonic neuronal aromatase, gender-specific primary cell cultures were prepared from embryonic day 15 mouse hypothalamus and cortex. Estrogen formation by cultured neurons was measured using an in vitro 3H2O product formation assay, and aromatase neurons were identified by immunocytochemistry using a highly specific antiserum. Aromatase activity (AA) per well and numbers of aromatase-immunoreactive (IR) neurons per microtubulus associated protein II-IR neurons x 10(5) were significantly higher in male hypothalamic cultures compared with female when grown in the absence of sex steroids. When AA was calculated per aromatase-IR neuron, no differences in enzyme activity were found between male and female. Therefore, the level of AA in individual male hypothalamic neurons is similar to the female, but a higher proportion of male neurons express aromatase. After T treatment, AA per well (P < or = 0.001) and AA/aromatase-IR cell (P < or = 0.005) in male and female hypothalamic cultures was significantly increased vs. controls. In addition, numbers of aromatase-IR neurons/microtubulus associated protein II-IR neurons x 10(5) were significantly higher after T exposure compared with controls (P < 0.001). Androgenic effects on hypothalamic AA and aromatase-IR cell numbers were dose-dependent and mediated via androgen receptor stimulation, since the observed effects were inhibited by the androgen-receptor antagonist flutamide. There was no effect of T on cortical AA or aromatase-IR cell numbers, indicating area-specific regulation of brain aromatase. We conclude that 1) sex differences in hypothalamic AA are due to a higher percentage of neurons expressing aromatase in males rather than to higher AA in individual male hypothalamic aromatase-IR cells, and 2) androgens influence the development of the fetal hypothalamic aromatase system. Because T influenced both the embryonic male and female hypothalamic neurons in culture, the developing mouse brain aromatase appears to be bipotential in response to androgen. The data suggest that environmental and genetic factors affecting androgen level and/or androgen receptor function in the developing brain could interfere with the sexual differentiation of estrogen forming neurons.