Estrogen Responsiveness of Normal Mouse Mammary Cells in Primary Cell Culture: Association of Mammary Fibroblasts with Estrogeiiic Regulation of Progesterone Receptors*

Abstract

Estrogens enhance proliferation of normal mouse mammary cells in vivo. When cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro reponsiveness to estrogen is not known. If cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional as measured by the ability of estrogens to regulate PgR were determined. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epitheilal cells responsiveness to estrogen. Collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultured (containing both epithelial cells and fibrolast) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quanitified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 [17-21-dimethyl-19-norpregna-4,9-diene-3-20-diene-3,20-diene] and 17.beta.-[3H]estrodiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 .+-. 0.8 .times. 10-10) and progestins (Kd = 3.3 .+-. 1.2 .times. 10-9 M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, cultures containing only fibroblasts which were derived by differential centrifugation were investigated. When 17.beta.-estradiol was added to the culture medium, a significant (P < 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain reponsiveness to estrogen in vitro, as measured herein, the epithelial derived by differential centrifugation and Percoll gradient sedimentation, did not. Estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. The presence of mammary fibroblast appears to be associated with epihelial cell responsiveness to estrogen in vitro.