Multiple structurally distinct ERα mRNA variants in zebrafish are differentially expressed by tissue type, stage of development and estrogen exposure

Abstract

It is well established that estrogen-like environmental chemicals interact with the ligand-binding site of estrogen receptors (ER) to disrupt transcriptional control of estrogen responsive targets. Here we investigate the possibility that estrogens also impact splicing decisions on estrogen responsive genes, such as that encoding ERα itself. Targeted PCR cloning was applied to identify six ERα mRNA variants in zebrafish. Sequencing revealed alternate use of transcription and translation start sites, multiple exon deletions, intron retention and alternate polyadenylation. As determined by quantitative (q)PCR, N-terminal mRNA variants predicting long (ERαL) and short (ERαS) isoforms were differentially expressed by tissue-type, sex, stage of development and estrogen exposure. Whereas ERαL mRNA was diffusely distributed in liver, brain, heart, eye, and gonads, ERαS mRNA was preferentially expressed in liver (female > male) and ovary. Neither ERαL nor ERαS transcripts varied significantly during development, but 17β-estradiol selectively increased accumulation of ERαS mRNA (~170-fold by 120 hpf), an effect mimicked by bisphenol-A and diethylstilbestrol. Significantly, a C-truncated variant (ERαS-Cx) lacking most of the ligand binding and AF-2 domains was transcribed exclusively from the short isoform promoter and was similar to ERαS in its tissue-, stage- and estrogen inducible expression. These results support the idea that promoter choice and alternative splicing of the esr1 gene of zebrafish are part of the autoregulatory mechanism by which estrogen modulates subsequent ERα expression, and further suggest that environmental estrogens could exert some of their toxic effects by altering the relative abundance of structurally and functionally distinct ERα isoforms.

Estrogen receptor 2b deficiency impairs the antiviral response of zebrafish

“…Interestingly, these observations resemble those of the arrested state of spermatogenesis described for adult zebrafish exposed to waterborne E2 (de Waal et al, 2009). As the indicated lack of feedback occurs at the E2 level, a late stage of the steroid synthesis pathway (Tokarz et al, 2013), an increase in the presence of its precursors, such as testosterone, is also to be expected.…”

“…Additionally, groups of larvae were exposed 2 dpf to E2 at final concentrations of 10 nM dissolved in DMSO (0.01%) (de Waal et al, 2009;Cotter et al, 2013). The exposure was continued until the end of the assay, refreshing 1/3 of the media per day.…”

“…The existing evidence concerning reproductive aspects of estrogenic endocrine regulation demonstrates the high susceptibility of zebrafish to exogenous hormonal disturbance, especially to those compounds of an estrogenic nature which are capable of promoting gonadal impairment and sex differentiation alterations (de Waal et al, 2009;Tokarz et al, 2013;Tokumoto et al, 2011). As most studies indicate, these effects involved profound alterations in the gene expression profile and the physiology of many key transcription factors whose genes display estrogen responsive elements in their regulatory regions (Baker and Hardiman, 2014;Larkin et al, 2003;Schiller et al, 2013;Urbatzka et al, 2012).…”

Cloning of multiple ERα mRNA variants in killifish (Fundulus heteroclitus), and differential expression by tissue type, stage of reproduction, and estrogen exposure in fish from polluted and unpolluted environments

“…However, one alternatively spliced ERα was cloned from killifish (ERαx), which was present in both SC and NBH populations (Greytak and Callard, 2007). Alternatively spliced ERα variants have been identified in many fish species (Cotter et al, 2013; Pakdel et al, 2000; Patino et al, 2000; Pinto et al, 2012; Seo et al, 2006; Tan et al, 1996; Xia et al, 1999). In particular, the expression of alternative long (ERα L , full-length) and short (ERα S , 5′-truncated) mRNA variants appears to be a conserved feature of teleosts (Cotter et al, 2013).…”

“…Alternatively spliced ERα variants have been identified in many fish species (Cotter et al, 2013; Pakdel et al, 2000; Patino et al, 2000; Pinto et al, 2012; Seo et al, 2006; Tan et al, 1996; Xia et al, 1999). In particular, the expression of alternative long (ERα L , full-length) and short (ERα S , 5′-truncated) mRNA variants appears to be a conserved feature of teleosts (Cotter et al, 2013). In rainbow trout, where the processing of the esr1 gene and production of alternative ERα S and ERα L isoforms was first described, the two variants have different estrogen binding and transactivation functions (Menuet et al, 2001; Pakdel et al, 2000).…”