Introduction
Estrogens are involved in the regulation of a wide range of processes in teleost reproduction (Lubzens et al, 2010; Schulz et al, 2010) and exert their functions mainly through ligand-activation of their specific cognate receptors. Two main classes of estrogen receptors have been reported in vertebrates: membrane-associated estrogen receptors (GPER), which are G-protein coupled receptors that mediate rapid nongenomic actions through intracellular signaling pathways (Filardo et al, 2012), and nuclear estrogen receptors (ESR), transcription factors that bind to estrogen response elements (EREs) on gene promoters to regulate their expression. (Tsai et al, 1994). Three Esr subtypes have been identified in most teleost species, Esr1, Esr2a (also named Esrβ1 or Esrβa), and Esr2b (also named Esrβ2 or Esrβb), of which Esr2a and Esr2b coding genes arose from the whole genome duplication event specific of the teleost lineage. Differences in binding affinity and seasonal expression patterns in reproductive tissues among estrogen receptor subtypes suggest different roles during gametogenesis along the brain-pituitary-gonad axis. Endocrine-disrupting compounds (EDCs) are known to act as agonists or antagonists of steroid receptor functions and interfere in the production of the receptors themselves. Thus, many different points in the endocrine control of fish reproduction can be potential targets for the actions of EDCs. In aquaculture, the presence of estrogenic compounds is a concern, both in water and in newly formulated commercial fish diets (Arpin-Pont et al, 2016; Nacher-Mestre et al, 2013;). This study aimed to investigate the role of the three nuclear estrogen receptor subtypes in European sea bass, assess the impact of endocrine-disrupting compounds on nuclear estrogen receptors using transactivation assays, generate in vitro stable clones, and evaluate their potential as bioassays tools for risk assessment.
Material and Methods
To investigate the role of the three nuclear estrogen receptors subtypes in European sea bass, analysis of the expression of their coding genes during a whole reproductive cycle was investigated by qPCR in testis, ovary and pituitary. Localization of the nuclear estrogen receptors along the brain-pituitary-gonad axis was performed by immunohistochemistry using specific antibodies for each subtype.
Using transactivation of an ERE-luciferase reporter gene assay, transient transfection was conducted in human embryonic kidney cell line (HEK293), to functionally characterize the three sea bass nuclear estrogen receptors. The effect of two possible EDCs, genistein – a phytoestrogen also present in fish meals – and fluoxetine – an antidepressant, mainly constituent of Prozac- were assessed across all Esr subtypes.
Based on results from transient transactivation assays, stable HEK293 clones expressing each Esr subtype were generated using antibiotic selection with G418 and Hygromycin. Clones were screened by treatments with 10^−8 M 17β-estradiol (E2), using ethanol as vehicle control. The most responsive clones were expanded and cryopreserved. Subsequent transactivation assays using E2 concentrations ranging from 10^−5 M to 10^−10 M were performed to determine the EC50 (half-maximal effective concentration) and Emax (the maximum response) values for each sea bass Esr subtype. Finally, to evaluate the potential of these stable clones as in vitro bioassay tools for EDCs risk assessment, environmental and biologically relevant samples (water, fish feed) were tested.
Results
The coding genes of the three nuclear estrogen receptor subtypes of sea bass are highly expressed in reproductive-related tissues such as pituitary and gonad. Quantitative analysis of esr1, esr2a and esr2b expression in the gonad and pituitary across the reproductive cycle revealed subtype and stage-specific expression patterns. Immunohistochemical localization along the pituitary-gonad axis confirmed differences among receptor subtypes depending on the gonadal stage.
Functional characterization using an ERE-luciferase reporter assay demonstrated distinct ligand affinities among the Esr subtypes, which translated into differential transactivation responses to the EDCs genistein and fluoxetine.
Furthermore, stable HEK293 clones expressing each sea bass Esr subtype were successfully generated and validated using dose-responses assays with E2 as natural ligand. Values for EC50 and Emax align with previous activations in transient transfections. Finally, the effect of different environmental and biological relevant samples on Esr subtypes confirm the reliability of these stable cell lines as in vitro bioassay tools for detecting and evaluating the activity of potential EDCs.
Conclusion
These findings indicate that the three nuclear estrogen receptors in European sea bass have non-redundant, subtype-specific roles in the regulation of gametogenesis. Consequently, exposure to EDCs—whether present in water or newly commercial fish feeds—may disrupt reproductive processes. Moreover, the development of stable Esr-expressing HEK293 clones offers a potential in vitro bioassay platform for the detection and risk assessment of EDCs in environmental and biological samples, with particular relevance to marine aquaculture.
Acknowledgements
Funded by MCIN/AEI/10.13039/501100011033/ and by ERDF a way of making Europe (PID2021-122929OB-C32) and by Generalitat Valenciana (CIAICO/2022/002). J.R. was supported by a research contract from “Programa Investigo” Generalitat Valenciana (INVEST/2023/560).
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