ESTROGEN RECEPTOR-BETA MESSENGER-RNA EXPRESSION IN RAT OVARY - DOWN-REGULATION BY GONADOTROPINS

We have examined the expression and regulation of the two estrogen rec eptor (ER alpha and ER beta) genes in the rat ovary, using Northern bl otting, RT-PCR, and in situ hybridization histochemistry. Northern blo tting results show that the ovary expresses both ER alpha and ER beta genes as single (similar to 6.5-kb) and multiple (ranging from similar to 1.0-kb to similar to 10.0-kb) transcripts, respectively. ER alpha mRNA is expressed at a level lower than ER beta mRNA in immature rat o varies. This relationship appears unchanged between sexually mature ad ult rats and immature rats. In sexually mature adult rats undergoing e ndogenous hormonal changes, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less constant with the exceptio n of the evening of proestrus when ER beta mRNA levels were decreased. Examination of ER beta mRNA expression at the cellular level, by in s itu hybridization, showed that ER beta mRNA is expressed preferentiall y in granulosa cells of small, growing, and preovulatory follicles, al though weak expression of ER beta mRNA was observed in a subset of cor pora lutea, and that the decrease in ER beta mRNA during proestrous ev ening is attributable, at least in part, to downregulation of ER beta mRNA in the preovulatory follicles, This type of expression and regula tion was not typical for ER alpha mRNA in the ovary. Although whole ov arian content of ER alpha mRNA was clearly detected by RT-PCR, no appa rent modulation of ER alpha mRNA levels was observed during the estrou s 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 localizat ion. To further examine the potential role of the preovulatory pituita ry gonadotropins in regulating ER beta mRNA expression in the ovary, w e used immature rats treated with gonadotropins. In rats undergoing ex ogenous hormonal challenges, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less unchanged after an inject ion of PMSG. In contrast, a subsequent injection of human CG (hCG) res ulted in a substantial decrease in whole ovarian content of ER beta mR NA. 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 tha n that observed for small and growing follicles. In addition, there is an abrupt decrease in ER beta mRNA expression in the preovulatory fol licles after hCG injection. The inhibitory effect of hCG on ER beta mR NA 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 o f hCG in down-regulating ER beta mRNA in cultured granulosa cells. Tak en together, our results demonstrate that 1) the ovary expresses both ER alpha and ER beta genes, although ER beta is the predominant form o f estrogen receptor in the ovary, 2) ER beta mRNA is localized predomi nantly to the granulosa cells of small, growing, and preovulatory foll icles, and 3) the preovulatory LH surge down-regulates ER beta mRNA. T hese results clearly implicate the physiological importance of ER beta in female reproductive functions.