Effects Of 17α-ethynylestradiol Exposure On Development And Reproduction Of Zebrafish And Its Corresponding Mechanisms

In recent years, the effects of environmental endocrine disrupting chemicals (EDCs) on the health of humans and wildlife have been a growing concern among researchers and policy makers. EDCs have the capacity to modulate the endocrine system and could possibly interfere with the reproductive system of wild life and even humans. The synthetic estrogen EE₂ is a common component of oral contraceptives. It enters the aquatic environment through domestic STWs. In two independent studies, concentrations of EE₂ in municipal STWs were reported to range from non-detectable to 7 ng/Lnd non-detectable to 42 ng/L A recent study conducted by the U.S. Geological Survey reported that 5.7% of rivers in the United States had EE₂ concentrations > 5 ng/L. Due to the long half-life of EE₂ and its bioconcentration in biota, the concentration of EE₂ in a fish body can be 332-fold higher than that in the environment and the potency of EE₂ can be 10- to 50-fold higher than that of E₂ and E₁ in vivo. Numerous studies have shown that fish exposed to EE₂ experienced vitellogenin (Vtg) induction, disrupted sex differentiation, and reproductive failure. Thus, EE₂ could potentially contribute to reproductive dysfunction in wild fish populations, given its high potency and wide distribution.To investigate the impact of EE₂ exposure on development and reproduction, zebrafish were exposed to 0.4, 2,10 ng/L EE₂ or solvent control from 2 dph to 90 dph. Fish were then transferred to clean water to recovery for 3 months. The mortality and pericardial edema rate in 2 ng/L and 10 ng/L EE₂-treated groups were significantly higher than that of the solvent control at 90 dph. The mean body weight and length of fish exposed to 10 ng/L EE₂ were also obviously lower than the control group. These results indicate there is a general toxic effect in exposure to concentration of EE₂ higher than 10 ng/L. We also investigated whether the Vtg mRNA expression could be induced in the first four weeks after hatch. RT-PCR indicates the Vtg mRNA could be induced earlier than 21 dph at 10 ng/l EE₂. The vtg-egfp transgenic zebrafish also shows a similar green fluorescent expression pattern at the same exposure period which is consistent with the RT-PCR results. Our findings suggest Vtg could be possibly used as a sensitive biomarker for estrogen even at early developmental stages.In the mechanism that EE₂ interferes with sex differentiation and development, the disrupted gonad development may be an important part. In this study, zebrafish were exposed to 0.4, 2 and 10 ng/L EE₂ or solvent control (ethanol) from 2 to 90 dph. Histological sections at 90 dph indicates a severe disruption of sex differentiation and development at 2 and 10 ng/L EE₂ in which the gonad of fish contained only immature ovary type tissue or even undifferentiated gonads. After a 3-month recovery period, growth and sex ratio were partially recovered. However, malformation of the sperm duct and reduced number of spermatozoa were still found in fish exposed to 2 ng/L and 10 ng/L EE₂. No obvious differences in gonad histological sections between EE₂ treated female fish and solvent control was found.Breeding studies at 180 dph revealed significant reproductive dysfunctions in all EE₂-treated groups. Female zebrafish produced fewer eggs and the egg viability at 12 hpf were lower than that of the control. To further investigate the respective effects of EE₂ on both sexes, we used a male/female replacement experiment. In the male replacement trial, female fecundity of EE₂-treated fish did not improve, but their egg viability had recovered. In the female replacement experiment, egg viability in EE₂-treated groups did not improve. However, female fecundity had partially recovered. Interestingly, a dose dependent decrease of fecundity was still found in these fish. Considering the results of sperm examination, these findings clearly indicate that EE₂ exposure impairs the reproductive functions of both male and female zebrafish.Some previous studies reported zebrafish exposed to endosulfan and nonylphenol (NP) resulted in disrupted primordial germ cells (PGCs) migration and distribution during embryo stages. However, no study has been carried out regarding the effects of EEs exposure on PGCs migration and distribution of zebrafish. In this study, we examined the effects of 10 ng/L and 100 ng/L EE₂ exposure on zebrafish PGC migration and distribution by labeling PGCs with GFP and in situ hybridization. The results were negative. We suspect the reason should be gonads at early stages are not sensitive to environmental estrogens. However, we can not exclude the possibility of adverse effects of other EEs on PGC.