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

Abstract

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.

Figure 1

Fluorescence images of zebrafish PGCs after exposure to various concentrations of EE2. A: In all treatment groups, normal PGCs were observed in the anterior region of the yolk extension at 24 hpf. B: In the 1 ng/L, 10 ng/L, and 100 ng/L EE2 exposure groups, ectopic PGCs were primarily observed along the branchial arch, similarly to the spadetail mutant. C: In the 500 ng/L, 1 μg/L and 2 μg/L EE2 exposure groups, many ectopic PGCs were observed along the branchial arch, on the back, in the abdomen, and along the trunk. A’, B’, and C’: Bright-field views show the morphology of the embryos.

Figure 2

Percentage of zebrafish embryos with ectopic PGCs in different exposure groups. In the control, 1 ng/L, 10 ng/L, and 100 ng/L EE2 exposure groups, less than 2% of the zebrafish embryos showed ectopic PGCs. In the 500 ng/L EE2 exposure group, approximately 13% of zebrafish embryos contained ectopic PGCs. In the 1 μg/L EE2 exposure group, approximately 20% of zebrafish embryos contained ectopic PGCs. In the 2 μg/L EE2 exposure group, approximately 19% of zebrafish embryos contained ectopic PGCs. These rates were significantly higher than that of other groups (p < 0.01, one-way ANOVA followed by Tukey’s test).

Figure 3

Effects of MO on GFP expression in 7 hpf embryos injected with recombinant plasmids. A, B: A mosaic pattern of GFP expression was detected throughout the embryos. C, D: GFP expression was undetectable after co-injection with MO and recombinant plasmids. A’, B’, C’, D’: Bright-field views show the morphology of the embryos.

Figure 4

Embryos with ectopic PGCs in different treatment groups. In tank water, ICI and MPP treatment induced ectopic PGC distribution in 14% and 10% of the embryos, respectively. PHTPP, esr2a-MO, and esr2b-MO treatment did not disrupt PGC distribution. An increased percentage of fish with ectopic PGCs at 24 hpf was observed after exposure to EE2 compared with exposure to tank water alone (i.e., without EE2). Approximately 37% and 40% of the embryos displayed ectopic PGCs after exposure to ICI and MPP, respectively. Fewer embryos showed ectopic PGCs expression after exposure to PHTPP, and esr2a expression was reduced by approximately 13% and 11%, respectively. Inhibition of esr2b expression did not affect PGC distribution.