ER transcriptional activity during menopause transition and aging

Molecular imaging, i.e. the non-invasive imaging of targeted macromolecules and of biological processes in living organisms is providing a novel, very powerful, technology for the progress of biomedical research by facilitating the study of physio-pathological events in living organisms and improving the discovery of novel drugs. The ERE-Luc mouse has been generated in 2001, this mouse is the first prototype of reporter animal in which a luciferase reporter gene driven by an estrogen-responsive promoter is expressed in all tissues. The gene encoding the firefly luciferase is a reporter gene, an enzyme that reacting with the substrate luciferine, causes the production of photons detectable quantitatively, by a charge-coupled device (CCD) camera. The use of in vivo imaging of the ERE-Luc mice provided us with the opportunity to study the physiological fluctuations of ERs in mice during estrous and to demonstrate that the state of ER transcriptionally activity is regulated by circulating estradiol only in reproductive organs and liver. In organ with not associated reproductive functions ERs are activated by hormones other than estradiol. We still do not have a clear understanding of the consequences of menopause on the state of activity of ERs in non reproductive organs. Our working hypothesis is that, after cessation of ovary functions, in non reproductive organs ERs might still be transcriptionally active due to a mechanism of unligated activation. Therefore, the principle objective of our work is the study of ER transcriptional activity at the menopause transition and during aging. Briefly, we quantified photon emission, in vivo, after systemic injection of the luciferase substrate luciferine, and luciferase activity by an in vitro enzymatic assay on tissue extracts as a measure of reporter gene expression. Furthermore, the data obtained from the quantification of luciferase activity were validated, by measuring extract the content of specific mRNA encoded by genes known to be direct targets of estrogens, in collected tissues. These experiments show how and the extent to which ER transcriptional activity changes from 6 up to 22 months of age. The addition of a group of ovx animals shows whether surgical ovx affects ER activity differently from natural menopause. The extent to which at menopause and after ERs are activated by different factors than estradiol is relevant for the understanding of the causal link between menopause and onset of disorders associated with aging.