G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish

Abstract

Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERβ), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERβ mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERβ mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels.

The Effects of 17α‐Ethinylestradiol on the Heart Rate of Embryonic Japanese Medaka (Oryzias latipes)

“…Few PPCPs have been investigated as extensively as EE2 from an environmental perspective, with many studies demonstrating high potency of EE2 to nontarget organisms (Parrott and Blunt 2005; Kidd et al 2007; Bhandari et al 2015). The toxicity of EE2 has often been attributed to ERα and ERβ activation, known as classical estrogen signaling (Jayasinghe and Volz 2012; Prossnitz and Barton 2014; Romano et al 2017). Exposure of EE2 to fish has led to the feminization of males and alterations in fertility and fecundity (Parrott and Blunt 2005; Kidd et al 2007).…”

“…These effects have been demonstrated in both the laboratory and a whole‐lake system (Parrott and Blunt 2005; Kidd et al 2007; Blanchfield et al 2015). More recently, there has been a greater interest in nonclassical estrogen signaling in nontarget organisms through GPER (Jayasinghe and Volz 2012; Romano et al 2017).…”

“…The toxicity of EE2 has been primarily attributed to the activation of ERα and ERβ, leading to alterations in the transcription of associated genes. This mode of action has been referred to as “classical estrogen signaling.” More recent evidence suggests that the G protein–coupled estrogen receptor (GPER, formerly known as GPR30) may play a role in the toxicity of estrogenic compounds in fish (Romano et al 2017). This receptor is localized intracellularly, on the membrane of the sarcoplasmic reticulum (Filardo and Thomas 2012; Prossnitz and Barton 2014; Barton 2016; Sharma et al 2018).…”

“…Expression of GPER ( gper1 ) occurs throughout the body of vertebrates, with higher levels found in the gonad, nervous system, and vasculature (Jayasinghe and Volz 2012). Recently, it has been demonstrated that 1 mg L −1 of E2 can induce tachycardia in embryonic zebrafish ( Danio rerio ; Romano et al 2017). It was also determined that GPER regulates heart rate independently of nuclear estrogen receptors, presenting a useful in vivo system for studying GPER activity (Romano et al 2017).…”

“…Recently, it has been demonstrated that 1 mg L −1 of E2 can induce tachycardia in embryonic zebrafish ( Danio rerio ; Romano et al 2017). It was also determined that GPER regulates heart rate independently of nuclear estrogen receptors, presenting a useful in vivo system for studying GPER activity (Romano et al 2017). Furthermore, it has been demonstrated that bradycardia is induced in embryonic zebrafish following exposure to 0.4 ng L −1 (Santos et al 2014).…”