Adaptive Significance of ERα Splice Variants in Killifish (Fundulus heteroclitus) Resident in an Estrogenic Environment

The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone-response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic-polluted environment (New Bedford Harbor, MA [NBH]) compared with those from a reference site overexpress estrogen receptor alpha (ERa) mRNA but are hyporesponsive to estradiol. Analysis of ERa mRNAs in the two populations revealed differences in splicing of the gene encoding ERa (esr1). Here we tested the transactivation functions of four differentially expressed ERa mRNAs and tracked their association with the hyporesponsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERa Delta 6 and ERa Delta 6-8 were specific to NBH killifish, had dominant negative functions in an in vitro reporter assay, and were heritable. Morpholino-mediated induction of ERa Delta 6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERaL) and short (ERaS) 5'-variants were similar transcriptionally but differed in estrogen responsiveness (ERaS >> ERaL). ERaS accounted for high total ER alpha expression in first generation (F1) NBH embryos/larvae but this trait was abolished by transfer to clean water. By contrast, the hyporesponsive phenotype of F1 NBH embryos/larvae persisted after long-term laboratory holding but reverted to a normal or hyper-responsive phenotype after two or three generations, suggesting the acquisition of physiological or biochemical traits that compensate for ongoing expression of negative-acting ERa Delta 6 and ERa Delta 6-8 isoforms. We conclude that a heritable change in the pattern of alternative splicing of ERapre-mRNA is part of a genetic adaptive response to estrogens in a polluted environment.