Alternative splicing of estrogen receptor alpha: potential mechanism for endocrine disruption and adaptation in teleost fishes

Abstract

Accumulating evidence from epidemiological, wildlife, and laboratory studies indicates that abnormalities of reproduction, development and physiology can be ascribed to environmental contaminants with biological activities. Many such contaminants disrupt essential hormone-regulated processes by virtue of their ability to interact with nuclear hormone receptors (endocrine disrupting chemicals, EDC). Of particular concern are chemicals that mimic or block estrogen signaling (xenoestrogens, XE) through their direct interaction with estrogen receptors (ER). The current model of XE action focuses on disrupted transcriptional activity, as measured by changes in the expression of ER-regulated genes. However, transcription is tightly coupled to splicing, by which a single target gene transcript is processed to multiple structurally and functionally different mRNAs. In theory, any XE that interacts with ER to regulate transcription has the potential to disrupt splicing, thereby affecting not only mRNA quantity but also quality. To address this hypothesis, alternative splicing of the gene encoding ER alpha (esr1), itself an estrogen responsive gene, was investigated. In these studies, killifish (Fundulus heteroclitus), an environmentally relevant species, and zebrafish (Danio rerio), an advantageous laboratory fish model, were used. First, the occurrence of ER alpha splice variants in adult tissues, in developing embryos and in response to estrogens in the two species was documented. Additionally, the effects of long-term, multigenerational XE exposure on ER alpha splicing were examined in two killifish populations, one from an estrogenic (polluted) site and a second population from a reference (unpolluted) site. A subset of ER alpha variants from killifish was expressed in cell culture to document their transcriptional activities. To determine the in vivo relationship between estrogen responsiveness and an ER alpha splice variant of interest, esr1 splicing was experimentally altered in living embryos by microinjecting morpholino oligonucleotides, and changes in induction of a panel of estrogen responsive gene targets were measured as markers of effect. These results provide evidence that dysregulation of mRNA processing is also a mechanism of XE action, and suggest that resultant ER alpha splice variants mediate the short-term effects of estrogen disruption and are also part of the adaptive response to long-term, multigenerational XE exposures in the natural environment.