The rate of prepubertal ductal morphogenesis as well as the incidence of hyperplastic growth in the adult mouse mammary gland is enhanced by 5 days of neonatal estradiol exposure. It is unknown whether estradiol acts through its nuclear receptor to affect growth in the neonatal gland. Immunohistochemical analysis indicates that estrogen receptor alpha (ER) is present in both the mammary epithelium and stroma before estradiol exposure is initiated at day 1. By 7 days of age, the frequency of ER-positive epithelial and stromal cells is significantly reduced in mammary glands of estradiol-exposed mice compared to controls. Coincident with this decrease in mammary ER is growth inhibition of the estradiol-exposed gland. At day 21, the exposed glands exhibit precocious epithelial outgrowth while significantly fewer stromal cells express ER compared to controls. By day 35, the exposed epithelium fills the fat pad while the exposed stromal cells express significantly more ER. To study the function of mammary ER, we have established mammary epithelial cell lines from oil and estradiol treated mice. Two cell lines from oil-treated and two from estradiol-exposed glands maintain ER expression in culture. Transient transfection of these ER-positive cell lines with a reporter vector containing an estrogen response element (ERE) demonstrates that the immunodetected ER is functional as a transcription factor in response to 100 nM estradiol. The in vitro transcriptional activity in response to estradiol is anti-estrogen sensitive, requires the presence of the ERE, and is independent of neonatal treatment in vivo. Our data indicate that neonatal estradiol exposure alters ER expression and mammary growth, but does not alter ER function.

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