Quantitative Analysis of Estrogen Receptor Proteins in Rat Mammary Gland* † Recipient of a Fellowship from Wenner-gren Foundation (sweden) and a Research Grant from Scandinavia Japan Sasakawa Foundation

Estrogen receptor a and b proteins (ERa and ERb) at various stages of development of the rat mammary gland were quantified by Western blotting. ERa and ERb 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 ERa content ranged from 0.30 ‐1.55 fmol/mg total protein (mean values). The ERb content of the same samples ranged between 1.06 ‐7.50 fmol/mg total protein. At every developmental stage, the ERb content of the mammary gland was higher than that of ERa. When receptor levels were normalized against b-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 ERb variant, originally called ERb2 but named ERbins in this paper, Western blots revealed that ERbins protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERbins paralleled those of the protein. Double immunohistochemical staining with anti-ERa and anti-ERbins antibodies revealed that ERbins protein colocalized with ERa in 70 ‐ 80% of the ERa-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERbins is regulated not only quantitatively, but also with regard to its cellular distribution. As ERbins acts as the dominant repressor of ERa, we suggest that its coexpression with ERa quenches ERa function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation. (Endocrinology 142: 3177‐3186, 2001)