Profile of estrogen-responsive genes in an estrogen-specific mammary gland outgrowth model. Mus musculus

Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching within the adipose stroma of the adolescent mouse mammary gland, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify the estrogen-responsive genes that are associated with estrogen-stimulated ductal elongation and branching in the mouse mammary gland in the absence of other ovarian hormones. We also wanted to determine if estrogen-responsive gene regulation at early stages of ductal elongation (ie. when ductal growth was minimal) was similar to those regulated after significant ductal elongation had occurred. To identify estrogen-regulated genes, ovariectomized prepubertal mice were exposed to 17beta-estradiol for four weeks, and mammary gland global gene expression analyzed by microarray analysis at various points during this time course. We determined that while many genes are regulated in all weeks of treatment, there remained a subset of genes that was uniquely regulated at each time-point. This observation was reflected in the biological functions of these genes; some categories were represented in all weeks of treatment while others were specific to only certain time-points. We have also identified estradiol-responsive genes in the mouse mammary gland that co-express with Estrogen Receptor alpha in human breast cancer, which may represent novel effectors of estrogen action and/or biomarkers for the progression of estrogen-dependent cancers and other estrogen-driven diseases. Overall design: For each time-course experiment, one frozen #4 mammary gland was individually pulverized from four to five animals per treatment group, then homogenized in 3mL Trizol (Invitrogen, Carlsbad, CA) and RNA was prepared according to the manufacturer’s protocol. The RNA from individual animals was then pooled for each treatment group (four to five animals per group) and further purified using the QIAGEN (Valencia, CA) RNeasy Mini kit (Cat. No. 74104) clean-up protocol. Gene expression analysis was conducted using Agilent Mouse Oligo arrays (pattern number 011978) (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer’s protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. In each case, samples from estradiol-treated animals were co-hybridized with the day 7 placebo sample. Due to the rapid increase in adiposity in the mammary fat pad in the ovariectomized placebo-treated mice in days 14 and 28, the day 7 placebo sample was used as a control for all estradiol treated samples to avoid any variation due to this biological difference. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (version 7.1) (Rosetta Biosoftware, Kirkland, WA).