Expression of estrogen receptor-beta protein in rodent ovary

Estrogen is an essential hormone for the LH surge and ovulation. The primar y 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 sy nthesize estrogen. To further elucidate the mechanisms mediating the action s 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 an d 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 estroge n-treated immature rats. Extracts from granulosa cells specifically bound a n estrogen response element and the complex was recognized by antisera to E R-beta. A synthetic steroid estrogen radioligand, [I-125]-17 alpha-iodoviny l-11 beta-methoxyestradiol ([I-125]-VME2), bound to cytosolic granulosa cel l preparations with high affinity (estimated K-D value of 401 +/- 83 pM, an d B-max 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. O verall, we have demonstrated that 1) ER-beta protein is the dominant estrog en receptor subtype present in rodent granulosa cells, 2) this receptor is functional, and 3) it is regulated by ovulatory doses of gonadotropins. Thu s, ER-beta is likely to be a mediator of estrogen action in rodent granulos a cells during follicular development.