The Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Departments of Biochemistry and Obstetrics and Gynecology, The University of Texas Southwestern Medical Center, 5323 Harry Mines Boulevard, Dallas, Texas 75235, USA. Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, USA.
J Neuroendocrinol. 1992 Feb;4(1):29-36. doi: 10.1111/j.1365-2826.1992.tb00341.x.
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.
在大鼠中，胎儿生命的最后一周下丘脑芳香酶活性显著但短暂增加。本研究旨在深入了解这种发育模式的调节。取包括正中基底下丘脑和视交叉上核（以下简称视前区）的下丘脑片段，在无血清培养基中培养 2 至 5 天。通过形成（3）H 2 O 来测量芳香酶活性，利用 [1β-（3）H]雄烯二酮或 [1β-（3）H]睾酮作为底物。在 0.3 μM [1β-（3）H]睾酮的饱和浓度下获得最大活性速率。用[1,2,6,7-（3）H]睾酮孵育后，通过将衍生的雌二醇重结晶至恒定比活度，证实了形成的[（3）H]雌二醇的身份。与之前的报道一致，在妊娠第 16 天（GD16）之前，体内下丘脑芳香酶活性可忽略不计，在 GD19 时显着增加（>5.0 pmol/h/mg 蛋白），此后在 GD22 时降低至低水平（~1.0 pmol/h/mg 蛋白）。在 GD17 之前进行移植的正中基底下丘脑-视前区片段在体外未能产生芳香酶活性。如果组织在 GD17 或 18 进行移植（即体内活性增加时），则酶活性不会继续增加，而是在体内观察到的下降之前维持两天。当组织在 GD19 进行移植时（即体内活性达到峰值时），观察到类似但立即下降。将外植体暴露于生长因子（胰岛素样生长因子 II、表皮生长因子和碱性或酸性成纤维细胞生长因子）或类固醇（雌二醇-17β、孕酮、睾酮、二氢睾酮和皮质酮）既不能在 GD19 之前增加芳香酶活性，也不能减轻其围产期下降。用离子载体 A23187 增加 Ca（2+）通量或激活环 AMP、环 GMP 或蛋白激酶 C 途径同样无效，血管紧张素 II 也是一种新提出的神经芳香酶刺激物。相比之下，激活环 AMP 途径（使用 forskolin 或 8-溴环 AMP）或雄激素睾酮和二氢睾酮可使芳香酶活性抑制 2-4 倍。这些结果表明：1）GD17 之前大鼠下丘脑芳香酶活性的出现需要展开下丘脑外事件，2）GD19 之前发生的芳香酶活性增加还需要下丘脑外输入，并且不涉及任何已知的细胞内信号转导途径，3）GD19 后观察到的活性下降在视前区内受到调节，并且似乎至少部分由环 AMP 形成的激活决定。讨论了雄激素的潜在作用。
