Estrogen Receptor-α knockout females, but not Estrogen Receptor-β knockout females, show male-specific liver gene expression

Estrogen protects females from hepatocellular carcinoma (HCC). To determine whether this protection is mediated by classic estrogen receptors, we tested HCC susceptibility in estrogen receptor-deficient mice. In contrast to a previous study, we found that diethylnitrosamine induces hepatocarcinogenesis to a much greater extent when females lack Esr1, which encodes Estrogen Receptor-α. Relative to wild-type littermates, Esr1 knockout females developed 9-fold more tumors. Deficiency of Esr2, which encodes Estrogen Receptor-β, did not affect liver carcinogenesis in females. Using microarrays and QPCR to examine estrogen receptor effects on hepatic gene expression patterns, we found that germline Esr1 deficiency resulted in the masculinization of gene expression in the female liver. For microarray analysis, 1.3 µg of total RNA from each sample was used for fluorescent probe preparation, using the Agilent Low Input Linear Amplification and Labeling Kit (Agilent Technologies Inc., Santa Clara, CA). Following linear amplification and dye labeling, the quality of samples was determined by measuring dye incorporation and cRNA concentration using the NanoDrop instrument. In the study examining the effect of deletion of Esr1 specifically in the liver, we examined gene expression in individual animals where probes were labeled with cyanine-3 (Cy-3) and the sex-reference control [pool of ½ B6 female (n=18) and ½ B6 male (n=15)] was labeled with cyanine-5 (Cy-5). In all other gene expression studies, hepatic RNA was prepared from individual mice and RNA pools were made for each group, with approximately the same amount of RNA from each individual, using Cy-5 to label probes and Cy-3 to label the sex-reference control (except for ERalpha WT Male-Rep2 and ERalpha KO Male-Rep2, for which the samples were labeled with Cy3 and the reference was labeled with Cy5). Hybridizations were carried out according to the manufacturer instructions, using the Agilent-014868 Mouse Whole Genome 4 x 44 Oligo Microarray G4122F (Agilent Technologies Inc., Santa Clara, CA), with 1.5 µg of labeled probe and 1.5 µg of labeled sex-reference control. The Agilent Microarray Scanner System G2565BA was used to measure hybridization signals and Agilent Feature Extraction software (version 9.1) was used for analysis.