Estrogen receptor null mice: What have we learned and where will they lead us?

Abstract

All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn. Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses. Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not. Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product. As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted. Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms. As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems. The αERKO mice continue to satisfy the confirmatory role of a knockout quite well. As summarized in Table 4, the phenotypes observed in the αERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ERα, including 1)the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland;2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male. The list of unpredictable phenotypes in the αERKO must begin with the observation that generation of an animal lacking a functional ERα gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type. The successful generation of βERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the αERKO. In support of this is our recent successful generation of double knockout, or αβERKO mice of both sexes. The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the αERKO, were quite surprising. In turn, certain estrogen pathways in the αERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus. Furthermore, it is apparent that several of the αERKO phenotypes may be aggravated by the downstream attenuation of progesterone and PRL action or even enhanced by increased androgen sensitivity, including those observed in the mammary gland, gonads, bone, and behavior. In addition, disruption of the ERα gene may have unmasked estrogen-signaling systems that were not easily detectable in the wild-type, such as 1) the apparent estrogen actions of the 4-OH-E2 in the αERKO uterus that are independent of ERα and ERβ; 2) the ability of estrogen to induce increased levels of hypothalamic PR in the αERKO female; 3) estradiol-induced potentiation of select neurons of the hippocampus; and 4) the protective effects of estrogen in the carotid vascular injury model. Finally, the concurrent descriptions of human mutations resulting in a lack of estrogen action due to a loss in ligand or functional receptor have illustrated the utility of the ER null mice as a model system to further understand the pathologies that result. Therefore, the αERKO mice have illustrated the several ways in which data collected from a knockout model can quickly contribute to the current knowledge concerning the function of a particular gene. However, it is worth noting the distinct differences in thought that occurred during the generation of the αERKO and βERKO models. At the time the work was initiated to generate the αERKO mice, much was known about the many roles of estrogen and the ER; therefore, several educated predictions of the possible phenotypes were possible and have since been confirmed, rejected, or reevaluated. However, the conception of the βERKO mice occured only 2 yr after the discovery of the ERβ. Because little was known about the function and role that the ERβ might play, it was difficult to make sound predictions. Therefore, the αERKO mice have played a significant role in confirming many of the roles thought to be fulfilled by estrogen, whereas the βERKO mice have and will continue to provide primary insight into the functions of the ERβ. The generation of both models, as well as a forthcoming description of the αβERKO, will prove invaluable in elucidating the precise roles fulfilled by each ER, as well as any possible cooperative roles the two receptors might play within the same tissue or even within the same cell.