Quantitative analysis of estrogen receptor proteins in rat mammary gland

Estrogen receptor alpha and beta proteins (ER alpha and ER beta) at various stages of development of the rat mammary gland were quantified by Western blotting. ER alpha and ER beta 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 ER alpha content ranged from 0.30-1.55 fmol/mug total protein (mean values). The ER beta content of the same samples ranged between 1.06-7.50 fmol/mug total protein. At every developmental stage, the ER beta content of the mammary gland was higher than that of ER alpha. When receptor levels were normalized against beta -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 ERP variant, originally called ER beta2 but named ER beta ins in this paper, Western blots revealed that ER beta ins protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ER beta ins paralleled those of the protein. Double immunohistochemical staining with anti-ER alpha and anti-ER beta ins antibodies revealed that ER beta ins protein colocalized with ER alpha in 70-80% of the ER alpha -expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ER beta ins is regulated not only quantitatively, but also with regard to its cellular distribution. As ER beta ins acts as the dominant repressor of ER alpha, we suggest that its coexpression with ER alpha quenches ER alpha function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation.