Estrogen receptor-beta mRNA expression in rat ovary: down-regulation by gonadotropins.
Estrogen receptor-beta mRNA expression in rat ovary: down-regulation by gonadotropins.
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DOI：
10.1210/mend.11.2.9887
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发表时间：
1997-02
影响因子：
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通讯作者：
M. Byers;G. Kuiper;J. Gustafsson;O. Park-Sarge
中科院分区：
文献类型：
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作者：
M. Byers;G. Kuiper;J. Gustafsson;O. Park-Sarge
我们用Northern印迹、RT-PCR和原位杂交组织化学方法研究了两种雌激素受体基因(ERα和ERβ)在大鼠卵巢中的表达和调控。Northern杂交结果表明，ERα和ERβ基因分别以单一(约6.5kb)和多个(约1.0kb到约10.0kb)转录本的形式表达。ERα在未成熟大鼠卵巢中的表达水平低于ERβ。这种关系在性成熟的成年大鼠和未成熟的大鼠之间似乎没有变化。在经历内源性激素变化的性成熟成年大鼠中，RT-PCR测定的整个卵巢ERβmRNA的含量基本保持不变，但发情前期晚上ERβmRNA水平下降除外。通过原位杂交在细胞水平检测ERβmRNA的表达，发现ERβmRNA在小卵泡、生长卵泡和排卵前卵泡的颗粒细胞中优先表达，但在黄体中有微弱的表达，发情后期ERβmRNA的减少至少部分归因于排卵前卵泡中ERβmRNA的下调。这种类型的表达和调控在卵巢中不是典型的ERαmRNA。虽然RT-PCR可以检测到整个卵巢ERαmRNA的含量，但在发情周期中没有观察到ERαmRNA水平的明显调节。通过原位杂交检测ERαmRNA在细胞水平的表达，发现ERαmRNA在整个卵巢低水平表达，没有特殊的细胞定位。为了进一步研究排卵前垂体促性腺激素在调节卵巢ERβmRNA表达中的潜在作用，我们使用了用促性腺激素治疗的未成熟大鼠。在接受外源性激素刺激的大鼠中，注射PMSG后，RT-PCR测定的整个卵巢ERβmRNA的含量基本保持不变。相反，随后注射人绒毛膜促性腺激素(HCG)导致整个卵巢ERβmRNA含量显著下降。ERβmRNA的原位杂交显示，小的、生长的和排卵前的卵泡在颗粒细胞中表达ERβmRNA。排卵前卵泡的ERβmRNA含量低于小卵泡和正在生长的卵泡。此外，注射hCG后，排卵前卵泡ERβmRNA的表达急剧下降。同时观察hCG对培养的颗粒细胞ERβmRNA表达的抑制作用。此外，刺激促黄体生成素/卵泡刺激素受体相关的细胞内信号通路的药物(Forskolin和佛波酯)很容易模仿hCG下调培养的颗粒细胞ERβmRNA的作用。综上所述，我们的结果表明：1)卵巢同时表达ERα和ERβ基因，尽管ERβ是卵巢中雌激素受体的主要形式，2)ERβmRNA主要定位于小的、生长的和排卵前期卵泡的颗粒细胞，以及3)排卵前的黄体生成素高峰下调ERβmRNA。这些结果清楚地暗示了ERβ在女性生殖功能中的生理重要性。
We have examined the expression and regulation of the two estrogen receptor (ER alpha and ER beta) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ER alpha and ER beta genes as single (approximately 6.5-kb) and multiple (ranging from approximately 1.0-kb to approximately 10.0-kb) transcripts, respectively. ER alpha mRNA is expressed at a level lower than ER beta mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ER beta mRNA levels were decreased. Examination of ER beta mRNA expression at the cellular level, by in situ hybridization, showed that ER beta mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ER beta mRNA was observed in a subset of corpora lutea, and that the decrease in ER beta mRNA during proestrous evening is attributable, at least in part, to down-regulation of ER beta mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ER alpha mRNA in the ovary. Although whole ovarian content of ER alpha mRNA was clearly detected by RT-PCR, no apparent modulation of ER alpha mRNA levels was observed during the estrous cycle. Examination of ER alpha mRNA expression at the cellular level, by in situ hybridization, showed that ER alpha mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ER beta mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ER beta mRNA. In situ hybridization for ER beta mRNA shows that small, growing, and preovulatory follicles express ER beta mRNA in the granulosa cells. The preovulatory follicles contain ER beta mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ER beta mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ER beta mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ER beta mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ER alpha and ER beta genes, although ER beta is the predominant form of estrogen receptor in the ovary, 2) ER beta mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ER beta mRNA. These results clearly implicate the physiological importance of ER beta in female reproductive functions.