BioProject

A proposed membrane-mediated mechanism of rapid non genomic response to estrogen has been the intense focus of recent research. More...

A proposed membrane-mediated mechanism of rapid non genomic response to estrogen has been the intense focus of recent research. Estren (Es), a synthetic steroid, is reported to act selectively through a rapid membrane-mediated pathway, rather than through the classical nuclear estrogen receptor (ER)-mediated pathway, to maintain bone density in ovaierectomized mice without uterotropic effects. To further evaluate the mechanism and physiological effects of Es we studied responses in adult ovariectomized mice. Keywords: estrogen receptor, nongenomic steriod action, uterus Overall design: Mice were treated with sesame oil vehicle (Sigma), E2 (Steraloids; 1 µg/mouse) estren (300 µg/mouse), or dihydrotestosterone (DHT, Steraloids; 50 µg/mouse) and uteri were collected 2 or 24 hours after treatment and snap frozen in liquid nitrogen. Three or four uteri from each treatment group were pooled and RNA was prepared using Trizol reagent (Invitrogen, Carlsbad CA) and the RNeasy clean up protocol (Qiagen Valencia CA). Gene expression profiling was conducted using Agilent Mouse Oligo arrays with ~20,000 genes represented (Agilent Technologies, Palo Alto, CA). Five hundred ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit according to manufacturer’s protocol. For each two color comparison, 750 ng of each Cy3- and Cy5-labeled cRNA were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven according to the Agilent 60-mer oligo microarray processing protocol prior to washing and scanning with an Agilent Scanner (Agilent Technologies, Wilmington, DE). Each sample pair was hybridized on 2 replicate chips. Data was obtained using the Agilent Feature Extraction software (v7.5, v8.1), using defaults for all parameters. Less...