Dataset Information

Cloning of multiple ER? 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.

ABSTRACT: To test the hypothesis that alternative splicing could be an adaptive mechanism for populations subject to multi-generational estrogenic exposures, we compared estrogen receptor alpha (ER?) splicing variants in two populations of killifish (Fundulus heteroclitus): one resident in an estrogenic polluted environment (New Bedford Harbor, NBH, MA, USA) and one from a relatively uncontaminated reference site (Scorton Creek, SC, MA, USA). In total we identified 19 ER? variants, each with deletions of one or more coding exons. Four of the variants with potential functional relevance were analyzed by qPCR to test for population differences in expression by tissue type, site, sex, seasonal reproductive status and estrogen treatment. Significantly, a 5'-truncated short form variant (ER?S) was highly expressed in liver and ovary, and was associated with seasonal reproductive activity in SC but not NBH fish. Both ER?S and the full-length long variant (ER?L) were estrogen-inducible (ER?S>ER?L) but the induction response was lower in NBH than in SC fish. In contrast, NBH killifish were hyper-responsive to estrogen as measured by expression of two other estrogen responsive genes: vitellogenin (Vtg) and aromatase B (AroB). Most strikingly, two ER? deletion variants (?6 and ?6-8), lacking ligand binding and activation function domains, were identified in a subset of NBH fish, where they were associated with reduced responsiveness to estrogen treatment. Together, these results support the hypothesis that alternative splicing of the esr1 gene of killifish could be an autoregulatory mechanism by which estrogen modulates the differential expression of ER?, and suggests a novel and adaptive mechanistic response to xenoestrogenic exposure.

SUBMITTER: Cotter KA

PROVIDER: S-EPMC4300264 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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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.

Cotter Kellie A KA Nacci Diane D Champlin Denise D Chuprin Jane J Callard Gloria V GV

Aquatic toxicology (Amsterdam, Netherlands) 20141218

To test the hypothesis that alternative splicing could be an adaptive mechanism for populations subject to multi-generational estrogenic exposures, we compared estrogen receptor alpha (ERα) splicing variants in two populations of killifish (Fundulus heteroclitus): one resident in an estrogenic polluted environment (New Bedford Harbor, NBH, MA, USA) and one from a relatively uncontaminated reference site (Scorton Creek, SC, MA, USA). In total we identified 19 ERα variants, each with deletions of ...[more]

PMID: 25550165

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Project description:Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine disrupting chemicals (EDCs) in the environment are responsible for developmental and reproductive abnormalities. We have previously described a killifish population resident in a highly polluted Superfund site (New Bedford Harbor, NBH) that shows evidence of exposure to an estrogenic environment and endocrine disruption. Here, we compare NBH with a local reference population (Scorton Creek, SC) for developmental patterns and direct effects of exogenous estradiol on the estrogenic markers, brain cytochrome P450 aromatase (CYP19A2 or AroB), hepatic vitellogenin (Vtg), and hepatic estrogen receptor alpha (ER alpha). In contrast to our previous observation of elevated ER alpha in NBH embryos, developmental levels of AroB and Vtg mRNAs did not differ between the two sites, demonstrating that not all estrogen-responsive genes are upregulated in NBH embryos. A dose-response experiment showed that NBH larvae are less responsive (lower maximum induction, as measured by ER alpha) and less sensitive (higher EC(50) for induction, as measured by AroB) to estradiol than SC larvae, changes that would be adaptive in an estrogenic environment. In contrast, induction of Vtg mRNA is similar in the two populations, indicating that the adaptive mechanism is target gene-specific. Based on the lower basal levels of ER alpha mRNA in several tissues from adult NBH fish vs SC fish (Greytak and Callard, 2007), we predicted estrogen hyporesponsiveness; however, induction of ER alpha by estradiol exposure in reproductively inactive males did not differ between the two sites. Moreover, AroB was more responsive and Vtg induction was greater (2d) or similar (5d) in NBH as compared to SC males. Worth noting is the high inter-individual variability in estrogen responses of gene targets, especially in NBH killifish, which may indicate evolving preadaptive or adaptive mechanisms. In conclusion, although multi-generational exposure to a highly polluted environment is associated with changes in basal levels of ER alpha mRNA, this is not a simple predictor of estrogen responsiveness. We hypothesize that adaptation of killifish to the estrogenic and polluted environment may be occurring through diverse mechanisms that are gene-, tissue type- and life-stage-specific.

Project description:The non-migratory killifish Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other "dioxin-like compounds" (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci (AHR1, AHR2, AHRR) in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor (MA, USA), a PCB Superfund site, and nearby reference sites.We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant FST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus.Together, these data suggest that F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.

Project description:Arsenic is a contaminant found worldwide in drinking water and food. Epidemiological studies have correlated arsenic exposure with reduced weight gain and improper muscular development, while in vitro studies show that arsenic exposure impairs myogenic differentiation. The purpose of this study was to use Fundulus heteroclitus or killifish as a model organism to determine if embryonic-only arsenic exposure permanently reduces the number or function of muscle satellite cells. Killifish embryos were exposed to 0, 50, 200, or 800 ppb arsenite (AsIII) until hatching, and then juvenile fish were raised in clean water. At 28, 40, and 52 weeks after hatching, skeletal muscle injuries were induced by injecting cardiotoxin into the trunk of the fish just posterior to the dorsal fin. Muscle sections were collected at 0, 3 and 10 days post-injury. Collagen levels were used to assess muscle tissue damage and recovery, while levels of proliferating cell nuclear antigen (PCNA) and myogenin were quantified to compare proliferating cells and newly formed myoblasts. At 28 weeks of age, baseline collagen levels were 105% and 112% greater in 200 and 800 ppb groups, respectively, and at 52 weeks of age, were 58% higher than controls in the 200 ppb fish. After cardiotoxin injury, collagen levels tend to increase to a greater extent and take longer to resolve in the arsenic exposed fish. The number of baseline PCNA(+) cells were 48-216% greater in 800 ppb exposed fish compared to controls, depending on the week examined. However, following cardiotoxin injury, PCNA is reduced at 28 weeks in 200 and 800 ppb fish at day 3 during the recovery period. By 52 weeks, there are significant reductions in PCNA in all exposure groups at day 3 of the recovery period. Based on these results, embryonic arsenic exposure increases baseline collagen levels and PCNA(+) cells in skeletal muscle. However, when these fish are challenged with a muscle injury, the proliferation and differentiation of satellite cells into myogenic precursors is impaired and instead, the fish appear to be favoring a fibrotic resolution to the injury.

Dataset Information

Cloning of multiple ER? 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.

Publications

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.

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