Characterization of estrogen non-responsive estrogen receptor alpha knock-in (ENERKI) mice

To distinguish between ligand-induced and ligand-independent estrogen receptor alpha (ERalpha) actions in vivo, we generated estrogen non-responsive ERalpha knock-in (ENERKI) mice. These mice have a mutation (glycine 525 to leucine, G525L) in the ligand-binding domain of ERalpha which significantly reduces ERalpha interaction with and response to endogenous estrogens, while not affecting growth factor activation of ligand-independent pathways. In addition, the synthetic ERalpha selective agonist propyl pyrazole triol (PPT) is still able to activate ligand-induced G525L ERalpha pathways. To produce the ENERKI mice, an ERalpha construct containing the G525L mutation was electroporated into ES cells, two independent positive clones were injected into mouse blastocysts to generate chimeras, and germline transmission was established.;ENERKI females were infertile due to anovulation. 89% of their ovaries contained large, hemorrhagic, cystic follicles. This physiology is consistent with a lack of estrogen negative feedback in the brain, which results in chronically elevated circulating levels of luteinizing hormone (LH). The ENERKI females had immature and hypoplastic uterine tissues and rudimentary mammary gland ductal trees. The similarity of the ENERKI and ERalpha knock-out (alphaERKO) mice phenotypes confirms ligand-induced activation of ERalpha is crucial in female reproductive tract development. Insulin-like growth factor-1 (IGF-1) treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, demonstrating ERalpha ligand-independent pathways are most likely active in these animals. Daily PPT treatments stimulated uterine and mammary gland ductal development and prevented formation of ovarian hemorrhagic cysts in adult ENERKI females.;ENERKI males were subfertile with normal efferent duct histology, unlike alphaERKO mice, which are infertile due to impaired efferent ductule fluid reabsorption. While 6- and 12-week-old ENERKI mice had normal testicular histology and sperm counts, 20- and 40-week-old males exhibited heterogeneous testicular atrophy and reduced sperm counts. These phenotypes indicate ENERKI males may have a defect in germ cell differentiation and confirms both ligand-induced and ligand-independent activation of ERalpha are crucial in male reproductive tract development. Long-term PPT treatments begun by day 4 restored the ability of ENERKI males to sire litters, while treatments begun at puberty were not successful, indicating neonatal ligand-induced ERalpha activation may be needed to induce normal adult male fertility rates.