Investigating the impact of estetrol and 17α-ethinylestradiol on thyroid hormonal signaling during zebrafish (Danio rerio) metamorphosis

The thyroid axis orchestrates key biological functions in fish, including metamorphosis. Disruption of thyroid signaling by endocrine disrupting compounds (EDCs), particularly estrogens, remains understudied during this sensitive period. This study compared the thyroidal effects of two estrogens: 17α-ethinylestradiol (EE2), a synthetic compound widely used in combined oral contraceptives (COCs), and estetrol (E4), a natural estrogen produced only during pregnancy and recently introduced as the estrogenic component of a new COC. Zebrafish (Danio rerio) were exposed to EE2 and E4 at concentrations ranging from 10 to 10,000x their respective measured (0.1 ng/L for EE2) or predicted (32 ng/L for E4) environmental levels from fertilization to 30 days post-fertilization (dpf). Samples were collected at 5 dpf for proteomic analysis to assess effects on thyroid organogenesis and early function, and at 14, 22, and 30 dpf to evaluate growth, thyroid histology, hormone levels, and transcriptomic profiles, thereby examining the effects on thyroid function throughout metamorphosis. Proteomic analysis at 5 dpf showed no disruption of thyroid organogenesis or function following exposure to either estrogen. However, both compounds induced concentration-dependent differentially expressed proteins (DEPs) linked to key developmental and metabolic pathways, with possible long-term effects on metamorphosis. DEPs were mainly associated with Rab signaling (RAB23, RAB35B, RAB38B), retinoic acid metabolism (CRABP1B, RDH5, RDH10A/B), mTOR signaling (RHEB, IGF1RB, BCL2A), oxidative stress (GPX9, TXNRD2.2, DAO.1/2, GNPAT), and energy metabolism (COX15, COX4I2). During metamorphosis, EE2 (≥ 100 ng/L) significantly reduced larval growth and thyroid signaling activity, as shown by modulation of thyroid-axis gene expression. In contrast, E4 did not affect growth or thyroid structure up to 320,000 ng/L, and triggered only modest transcriptomic changes in thyroid-axis genes at 32,000 ng/L. This study represents the first comparative assessment of EE2 and E4 on fish thyroid using a multiparametric approach at early and later developmental stages. These findings demonstrate that while both compounds influenced early developmental and metabolic pathways, only EE2 disrupted thyroid signaling during metamorphosis and induced phenotypic impairments. E4 caused weaker effects on the thyroid axis and did not induce any observable metamorphic disruptions. Overall, E4 appears to pose a lower environmental risk despite its early proteomic impact.