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. Nuclear estrogen receptors are transcription factors that bind to estrogen response elements (EREs) on gene promoters to regulate their expression (Tsai et al, 1994), and three subtypes of these receptors have been identified in teleosts. Differences in binding affinity and seasonal expression patterns in reproductive tissues among estrogen receptor subtypes suggest different roles during oogenesis, vitellogenesis and testicular development along the brain-pituitary-gonad axis. It is known that endocrine-disrupting compounds (EDCs) may act as agonists or antagonists of regulatory actions of steroid receptors or of 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 in water is as worrying as its presence in new formulation of commercial fish diets (Arpin-Pont et al, 2016; Nacher-Mestre et al, 2013; Quesada-García et al, 2012). Nevertheless, the lack of information available on the role of each nuclear estrogen receptor in teleosts, including European sea bass, makes it difficult to have an accurate knowledge on the impacts of EDCs on reproduction. This study focused on investigating the role of the three nuclear estrogen receptor subtypes in European sea bass, on evaluating the impacts of endocrine-disrupting compounds on nuclear estrogen receptors functions and on their use as 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 the human embryonic kidney cell line HEK293, we have performed transient transfections (1) to characterize nuclear estrogen receptors (Esr1, Esr2a and Esr2b) by using transactivation of an ERE- luciferase reporter gene assay; (2) to study the effect of two possible EDCs, genistein – a phytoestrogen also present in fish meals – and fluoxetine – an antidepressant, mainly constituent of Prozac - on all nuclear estrogen receptors from sea bass.
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. Quantification of esr1, esr2a and esr2b expression in the gonad and pituitary during a whole reproductive cycle showed different expression patterns depending on stage and subtype. Localization of the three nuclear estrogen receptors along the pituitary-gonad axis showed differences among subtypes depending on the gonadal stage.
The functional characterization of the nuclear estrogen receptors showed that there are different ligand affinities among the nuclear estrogen receptor subtypes, which also translate into differential receptor responses when we evaluate the effect of potential EDCs, such as genistein and fluoxetine.
Conclusion
The results suggest that the three nuclear estrogen receptors of European sea bass are not redundant and have differential roles in the regulation of gametogenesis, as proposed also in other teleosts, which means that the effects produced by EDCs can induce potential adverse effects at reproductive level.
The results also show that in vitro bioassays using nuclear estrogen receptors are a good tool for risk assessment of potential endocrine-disrupting compounds.
Acknowledgements
Supported by funds from MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22). PISP. received Portuguese national funds from FCT (Foundation for Science and Technology) through project UIDB/04326/2020 and researcher contract DL57/2016/CP1361/CT0015 with the Univ.Algarve.
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