Molecular characterization of the uterotrophic assay: temporal gene expression changes in uterus and pituitary gland following ethinylestradiol.

The uterotrophic bioassay is a recognized tool for assessing estrogenic activities of chemicals, relying on estrogen-induced uterine growth. This study employed the assay in immature female rats using ethinyl estradiol (EE) to explore temporal gene expression dynamics in the uterus and pituitary gland, both estrogen-responsive organs. Gene expression changes were evident as early as 2 h post-EE administration in the uterus, with early responses indicating increased proliferation, macromolecule synthesis, and immune responses-notably through WNT/β-catenin signaling and cholesterol biosynthesis. Estrogen's influence on inflammation was observed, with macrophage polarization shifting from anti-inflammatory M2 to pro-inflammatory M1 types over time. In the pituitary gland, early gene upregulation suggested proliferative and metabolic responses to estrogen. The findings enhance understanding of estrogen's effects on uterine and pituitary function during the uterotrophic assay, offering avenues for assay optimization, improved in vitro models, and refined adverse outcome pathways (AOPs).