Use of antagonists and morpholinos in loss-of-function analyses: estrogen receptor ESR2a mediates the effects of 17alpha-ethinylestradiol on primordial germ cell distribution in zebrafish

BACKGROUND Various chemicals released into the aquatic environment adversely affect the reproductive system of fish, particularly by changing gonad structure and function. 17alpha-ethinylestradiol (EE2) is a potent environmental estrogen that disrupts sexual differentiation and normal reproduction in fish. Previous studies have shown that exposure to endocrine-disrupting chemicals (EDCs) disrupts the migration of primordial germ cells (PGCs) in zebrafish. METHODS To investigate the effects of EE2 exposure on PGC migration, zebrafish embryos were injected with gfp-nanos mRNA to label PGCs and subsequently exposed to different concentrations of EE2. Typical estrogen receptor antagonist treatment and morpholino knockdown experiments were used to identify functional estrogen receptors that mediate the effects of EE2. RESULTS The migration of PGCs was disrupted after exposure to high concentrations of EE2 (1 mirog/L). Loss-of-function analyses were performed for estrogen receptor ESR1, ESR2a, and ESR2b, and only loss of ESR2a resulted in a decreased number of ectopic PGCs following exposure to 1 mirog/L EE2. CONCLUSIONS EE2 exposure disrupts PGC migration and distribution, and this effect is mediated through the estrogen receptor ESR2a.