Estrogen receptor α and β proteins (ERα and ERβ) at various stages of development of the rat mammary gland were quantified by Western blotting. ERα and ERβ recombinant proteins were used as standards, and their molar concentrations were measured by ligand binding assays. In 3-week-old pregnant, lactating, and postlactating rats the ERα content ranged from 0.30-1.55 fmol/μg total protein (mean values). The ERβ content of the same samples ranged between 1.06-7.50 fmol/μg total protein. At every developmental stage, the ERβ content of the mammary gland was higher than that of ERα. When receptor levels were normalized against β-actin, it was evident that ER expression changed during development, with maximum expression of both receptors during the lactation period. With an antibody raised against the 18-amino acid insert of the ERβ variant, originally called ERβ2 but named ERβins in this paper, Western blots revealed that ERβins protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERβins paralleled those of the protein. Double immunohistochemical staining with anti-ERα and anti-ERβins antibodies revealed that ERβins protein colocalized with ERα in 70-80% of the ERα-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERβins is regulated not only quantitatively, but also with regard to its cellular distribution. As ERβins acts as the dominant repressor of ERα, we suggest that its coexpression with ERα quenches ERα function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation.
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