Identification of hormone interactions important for mammary gland proliferation and gene expression

Using our unique and authentic animal model, the pig, we determined which genes in the normal mammary gland are regulated in response to different combinations of the ovarian hormones estrogen and progesterone and the pituitary hormone prolactin. We surgically removed ovaries from 32 pigs to deplete endogenous estrogen and progesterone, and administered bromocriptine to suppress endogenous prolactin. Pigs were treated with all combinations of estrogen, progesterone and bromocriptine and/or haloperidol (to induce prolactin secretion), in a factorial design. Affymetrix arrays were used to analyze RNA from the mammary glands of these pigs and we found that estrogen, prolactin and the interaction of estrogen*prolactin were the primary regulators of gene expression in the mammary gland. This is in contrast to rodents where progesterone has an important role in ductal elongation and branching. Functional enrichment analyses suggest that estrogen promotes cell division while prolactin stimulates elements of fatty acid metabolism and an inflammatory response (in addition to milk protein genes). These results concur with phenotypic changes where estrogen, prolactin and estrogen*prolactin were the most important regulators of morphological development, cell proliferation and steroid receptor expression. Regression analysis of expression data against the rate of cell proliferation in all 32 pigs identified 1669 genes correlated with proliferation (FDR a=0.01). Regression analysis of expression data against proliferation in the 16 pigs treated with E identified 71 genes correlated with proliferation. 29 genes were common to both lists and also regulated by estrogen, and of these 16 have known functions in the cell cycle while the other 13 have never been associated with estrogen regulated mammary gland growth or cell proliferation. Twenty six of these genes were more highly correlated with proliferation than MKI67 (Ki67). Thirty six porcine mammary gland RNA samples were analyzed. Four were from sham-operated saline treated controls. All hormone treatment combinations were given to four pigs