Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption

Abstract

Endocrine-disrupting chemicals are known to impact multiple hormonal axes of vertebrates, among which the thyroid system is crucial for multiple developmental and physiological processes. Thus, the present study focused on the semi-quantitative visualization of intrafollicular triiodothyronine (T3) and thyroxin (T4) in zebrafish embryos as a potential test system for the detection of disrupted thyroid hormone synthesis. To this end, an antibody-based fluorescence double-staining protocol for whole-mount zebrafish embryos and larvae was adapted to simultaneously detect intrafollicular T3 and T4. During normal development until 10 days post-fertilization (dpf), the number of thyroid follicles increased along the ventral aorta. Concentrations of T4 and T3, measured by fluorescence intensity, increased until 6 dpf, but decreased thereafter. Exposure of zebrafish embryos to propylthiouracil (PTU), a known inhibitor of TH synthesis, resulted in a significant decrease in the number of follicles that stained for T3, whereas a trend for increase in follicles that stained for T4 was observed. In contrast, fluorescence intensity for both thyroid hormones decreased significantly after exposure to PTU. Overall, the zebrafish embryo appears to be suitable for the simultaneous visualization and detection of changing intrafollicular TH contents during normal development and after PTU treatment.

The Role of Estrogen and Thyroid Hormones in Zebrafish Visual System Function

“…At ~72 hpf, endogenous TH synthesis begins (Porazzi et al, 2009;Darras et al, 2011;Gothie et al, 2019;Vancamp et al, 2019) causing internal TH levels to increase significantly, peaking at 10 dpf (T3) and 21 dpf (T4) (Chang et al, 2012). A more recent study measuring T3 and T4 levels using fluorescent antibodies found hormone levels peak earlier, at 6 dpf, and then decrease (Rehberger et al, 2018). Prior to hatching, thyroglobulin expression begins at 32 hpf and Na + /I + symporter expression starts at 40 hpf (Alt et al, 2006).…”

AOP Report: Thyroperoxidase Inhibition Leading to Altered Visual Function in Fish Via Altered Retinal Layer Structure

“…Inhibition of TPO is usually measured in in vitro assays using mammalian source materials (Friedman et al, 2016). Nevertheless, a number of studies have directly quantified T4 present in the thyroid follicles to show that model TPO inhibitors such as the pharmaceuticals PTU and methimazole (a model TPO inhibitor) reduce TH synthesis in zebrafish (Raldúa & Babin, 2009;Rehberger et al, 2018;Thienpont et al, 2011). Studies in rats (Cooper et al, 1983(Cooper et al, , 1984 and African clawed frog (Tietge et al, 2010) further support the presence of this relationship across vertebrates.…”

“…In fish early life stages, whole-body TH level measurements are often used as a proxy for serum TH levels, because obtaining serum is practically not feasible. Time-, dose-, and incidence-concordance can be derived from the combined studies of Rehberger et al (2018) and Thienpont et al (2011) showing reduced TH synthesis after treatment with PTU and methimazole, as well as Stinckens et al (2020), If Yes, levels of confidence have been taken from the previously reviewed AOP 159.…”

Composition and endocrine effects of water collected in the Kibale national park in Uganda

“…In addition, endpoints included in a General Developmental Score (GDS) have been associated with developmental toxicity caused by thyroid disruptors, including abnormal iridophore pigmentation, beat and glide swimming and resorption of the yolk sac (Jomaa et al, 2014). Finally, given the aforementioned conservation of thyroid development in teleosts, early detection of thyroid disruption is possible using whole-mount intrafollicular TH staining which could potentially be implemented into current OECD zebrafish assays (Rehberger et al, 2018). Interestingly, a novel fluorescent transgenic line expression under the control of the zebrafish thyroglobulin promoter allows for in vivo monitoring of thyroid gland development.…”