Fitzpatrick S L, Funkhouser J M, Sindoni D M, Stevis P E, Deecher D C, Bapat A R, Merchenthaler I, Frail D E
Molecular Biology Division, Women's Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087, USA.
Endocrinology. 1999 Jun;140(6):2581-91. doi: 10.1210/endo.140.6.6928.
Estrogen is an essential hormone for the LH surge and ovulation. The primary source of estrogen is from ovarian granulosa cells and in rats, estrogen, in turn, increases granulosa cell number and enhances FSH-stimulated gene expression in these cells. Thus, rat granulosa cells both respond to and synthesize estrogen. To further elucidate the mechanisms mediating the actions of estrogen in granulosa cells, we have identified and characterized the estrogen receptor-beta (ER-beta) subtype in rodent granulosa cells. ER-beta protein was localized to the nuclei of rat granulosa cells in preantral and antral follicles by immunocytochemistry, coincident with the location of ER-beta messenger RNA (mRNA). Immunoprecipitation and Western blot analysis using ER-beta specific antisera demonstrated a protein of approximately 60 kDa in granulosa cells prepared from PMSG-primed immature mice and estrogen-treated immature rats. Extracts from granulosa cells specifically bound an estrogen response element and the complex was recognized by antisera to ER-beta. A synthetic steroid estrogen radioligand, [125I]-17alpha-iodovinyl-11beta-methoxyestradiol ([125I]-VME2), bound to cytosolic granulosa cell preparations with high affinity (estimated K(D) value of 401 +/- 83 pM, and Bmax value of 102 +/- 9 fmol/mg protein). ER-beta protein levels rapidly declined following hCG treatment consistent with the reported decrease in binding activity and ER-beta mRNA levels by high levels of gonadotropins. Overall, we have demonstrated that 1) ER-beta protein is the dominant estrogen receptor subtype present in rodent granulosa cells, 2) this receptor is functional, and 3) it is regulated by ovulatory doses of gonadotropins. Thus, ER-beta is likely to be a mediator of estrogen action in rodent granulosa cells during follicular development.
雌激素是促黄体生成素峰和排卵所必需的激素。雌激素的主要来源是卵巢颗粒细胞，在大鼠中，雌激素反过来会增加颗粒细胞数量，并增强这些细胞中促卵泡激素刺激的基因表达。因此，大鼠颗粒细胞既能对雌激素作出反应，也能合成雌激素。为了进一步阐明介导雌激素在颗粒细胞中作用的机制，我们已经在啮齿动物颗粒细胞中鉴定并表征了雌激素受体β（ER-β）亚型。通过免疫细胞化学方法，ER-β蛋白定位于大鼠窦前卵泡和窦卵泡颗粒细胞的细胞核中，这与ER-β信使核糖核酸（mRNA）的定位一致。使用ER-β特异性抗血清进行免疫沉淀和蛋白质印迹分析表明，从经孕马血清促性腺激素（PMSG）预处理的未成熟小鼠和经雌激素处理的未成熟大鼠制备的颗粒细胞中，存在一种约60 kDa的蛋白质。颗粒细胞提取物特异性结合雌激素反应元件，并且该复合物可被抗ER-β血清识别。一种合成类固醇雌激素放射性配体，[125I]-17α-碘乙烯基-11β-甲氧基雌二醇（[125I]-VME2），以高亲和力结合到细胞质颗粒细胞制剂上（估计解离常数K(D)值为401±83 pM，最大结合容量Bmax值为102±9 fmol/mg蛋白质）。人绒毛膜促性腺激素（hCG）处理后，ER-β蛋白水平迅速下降，这与高水平促性腺激素导致结合活性和ER-β mRNA水平下降的报道一致。总体而言，我们已经证明：1）ER-β蛋白是啮齿动物颗粒细胞中存在的主要雌激素受体亚型；2）该受体具有功能；3）它受排卵剂量促性腺激素的调节。因此，在卵泡发育过程中，ER-β可能是雌激素在啮齿动物颗粒细胞中发挥作用的介质。
