Yuan H, Bowlby D A, Brown T J, Hochberg R B, MacLusky N J
Division of Reproductive Science, Toronto Hospital Research Institute, Ontario, Canada.
Endocrinology. 1995 Jan;136(1):96-105. doi: 10.1210/endo.136.1.7828562.
In vitro autoradiographic methods have been developed for selective measurement of occupied and unoccupied estrogen receptors (ERs) in brain tissue sections. Addition of protamine sulfate traps unoccupied ERs in the tissue sections, allowing them to be detected after a short period of incubation with labeled estrogen. Occupied ERs are assessed, after washing in buffer without protamine to eliminate unoccupied receptor, by incubating the sections for 2 h at 37 C to exchange isotopically labeled steroid for the endogenous unlabeled ligand. Total ER binding capacity is estimated by summing the values for occupied and unoccupied ER. In all brain regions of normal females, ER occupation is low at estrus, reflecting the very low levels of circulating estradiol present at this stage of the estrous cycle, rising to approximately 50% of binding capacity at proestrus. By contrast, in intact males ER occupation varies considerably between brain regions, from a high of 55% of binding capacity in the bed nucleus of the stria terminalis to a low of 21% in the hypothalamic arcuate nucleus. Gonadectomy or treatment of intact males with the aromatase inhibitor 4-hydroxy androstenedione greatly reduces or eliminates ER occupation, depending on the brain region. In both sexes, changes in levels of endogenous gonadal steroids have little effect on total (occupied plus unoccupied) ER concentrations, with the exception of the hypothalamic ventromedial nucleus of the female, in which total ER concentration declines at estrus. These results are consistent with the hypothesis that local aromatization may be the primary determinant of regional ER occupation in the brain of the male rat, in contrast to the female, in which high levels of ER occupation are found only during the preovulatory estrogen surge. Although physiological changes in circulating estradiol and aromatizable androgen concentrations induce large changes in ER occupation, they have little effect on total ER content in most regions of the brain, suggesting that previous reports of changes in ER messenger RNA levels under different conditions of gonadal steroid exposure may not be directly reflected in steady state levels of the cognate receptor site.
已经开发出体外放射自显影方法，用于选择性测量脑组织切片中已占据和未占据的雌激素受体（ERs）。添加硫酸鱼精蛋白可捕获组织切片中未占据的ERs，使其在与标记雌激素短时间孵育后能够被检测到。在不含鱼精蛋白的缓冲液中洗涤以消除未占据的受体后，通过将切片在37℃孵育2小时，以用同位素标记的类固醇交换内源性未标记的配体，来评估已占据的ERs。通过将已占据和未占据的ER的值相加来估计总ER结合能力。在正常雌性的所有脑区中，发情期的ER占有率较低，反映出发情周期这个阶段循环雌二醇水平非常低，在动情前期上升至结合能力的约50%。相比之下，在完整雄性中，ER占有率在不同脑区之间差异很大，从终纹床核中高达结合能力的55%到下丘脑弓状核中低至21%。去势或用芳香化酶抑制剂4-羟基雄烯二酮处理完整雄性，根据脑区不同，会大大降低或消除ER占有率。在两性中，内源性性腺类固醇水平的变化对总（已占据加未占据）ER浓度影响很小，但雌性的下丘脑腹内侧核除外，其中总ER浓度在发情期下降。这些结果与以下假设一致，即与雌性相反，局部芳香化可能是雄性大鼠脑中区域ER占有率的主要决定因素，在雌性中，只有在排卵前雌激素激增期间才发现高ER占有率。尽管循环雌二醇和可芳香化雄激素浓度的生理变化会引起ER占有率的大幅变化，但它们对大脑大多数区域的总ER含量影响很小，这表明先前关于在不同性腺类固醇暴露条件下ER信使RNA水平变化的报道可能不会直接反映在同源受体位点的稳态水平上。
