Human estrogen receptor beta-specific monoclonal antibodies: Characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues

Investigation of the role of the second, more recently described estrogen receptor, denoted ERβ, will be critical in understanding the molecular mechanisms underlying tissue-specific gene regulation by estrogens. Expression of ERβ in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization and size of the endogenous ERβ protein, due, in part, to the limited availability of human ERβ-specific antibodies. Thus, our aim was to generate specific antibodies to human ERβ and use them to determine the tissue-specific distribution and size(s) of the ERβ protein. To this end, we have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ERβ. The antibodies, made in mice against human ERβ amino acids 256-505 (hormone binding domain lacking the F domain), are designated CFK-E12 (E12), CMK-A9 (A9) and CWK-F12 (F12) and were determined to be the IgG γ1 isotype for E12, and IgG γ2b for A9 and F12. All three monoclonal antibodies could be used to detect in vitro translated, baculovirus expressed, and cell transfected and expressed ERβ protein by Western blot analyses, and all failed to detect ERα. A9 and F12 were able to immunoprecipitate efficiently the native form of ERβ protein in the presence and absence of estradiol. Epitope mapping studies indicate that the E12 and F12 antibodies recognize overlapping peptide sequences in the N-terminal region of the hormone-binding domain, a region that is highly conserved among species. Immunocytochemical studies with these antibodies reveal nuclear-specific localization of the ERβ protein in granulosa cells of the rat ovary. Nuclear ERβ is also specifically localized in epithelial and some stromal cells of mouse and rat epididymis. Western blot analysis with protein extracts from ovarian granulosa cells of human, rat, mouse, and pig showed a ca. 52 kDa and an additional ca. 62-64 kDa band in these species. These results indicate the presence of two predominant molecular size forms of the ERβ protein in ovarian granulosa cells and demonstrate the utility of these antibodies for detection of ERβ in the human and in several other mammalian species.