Mechanisms Of EE₂ Damage Ovary, Liver And Gill In Adult Female Zebrafish（Danio Rerio）

Environmental estrogens（EE）, bring great influence to the fish, were widely exists in natural waters.Previous researches mainly focused on the impact of environmental estrogens on the early embryonic development and male gonadal sex differentiation of fish, while whether the EE effect on female ovarian development remained unknown.In order to shed light on the possible impact of EE on fish ovarian development and oogenesis, in the present study, 17α-Ethynylestradiol（EE₂）, a typical EE was administered to the zebrafish（Danio rerio） female adults by 6-day exposure. The induction ofera andvtgexpressions in the liver proved the strong estrogenic effect of EE₂ on zebrafish female adults.Histological result showed that, though the ovarian development and oogenesis of fish in low-dose group（20 ng/L EE₂） were found normal, the oocyte content of mid-vitellogenic stage（MV） lost, full-grownstage（FG） oocytes cell proceeded into atresia in the ovary of high-dose group（100 ng/L EE₂）.To elucidate the possible molecular mechanism involved, expressions ofgenes participated in sex steroid synthesis in brain-pituitary-gonadal axis were analyzed by quantitative PCR method（qRT-PCR）, as well as the plasma 17b-estrodial（E₂） level and vtg expression in the liver.qRT-PCR result revealed that EE₂（100 ng/L） significantly downregulated the expressions of cyp19a1 b, gnrh-II and gnrhr-II, which might possibly suppress the synthesis of pituitary stimulating follicle-stimulating hormone（FSH）, and luteinizing hormone（LH）. Consequently, in the pituitary, it was found that the expression of fshβ and lhβ was significantly down-regulated, suggesting decreasing FSH and LH level. Moreover, expressionsof fshr 、lhr（receptorsof FSH and LH in the ovary） were also significantly down-regulated, which might probably inhibit the expressions of cyp19a1 aand 20b-hsd-II, genes encoding enzymes responsible for E₂ and 17a, 20b, dihydroxy-4-pregnen-3-one（DHP） production, respectively, which was inconsistent with our results. In accordance to our previous founding, the plasma E₂ level was dramatically decreased after EE₂（100 ng/L） exposure. Though the level of plasma DHP was not measured, the sharply decreased 20b-hsd-II mRNA level will inevitably lead to declining plasma DHP level. In addition, except for decreased plasma E₂ level, the expression of era in the ovarywas also significantly decreased. The down-regulated eraexpression, together with the decreased E₂ level, might lead to decreasingyolk accumulation in oocytes, resulting in content loss in MV stage oocytes. On the other hand, thepossible insufficient synthesis of DHP might lead to the competence loss of FG oocyte and the subsequent apoptosis（atresia） thereafter. All these results were inconsistent with the founding of histology. In summary, we strongly suggest that, 100 ng/L EE₂ exposure lead to ovarian dysplasia and inhibition of oogenesis in female zebrafish possibly through the suppression of genes responsible for estrogen and progesterone synthesis and regulations, and subsequently decreased E₂ and DHP levels.Except for the ovarian dysplasia and inhibition of oogenesis, bleached body color, erected scales, gill hyperemia, abdominal dropsy, swelled liver and enlarged heart size were also observed in the zebrafish exposed to high-dose of EE₂, suggesting great impairment of liver and gill in the fish. Consequently, the damage of fish liver and gill was analyzed by histology.Histological results revealed that, though no obvious physiological change was observed in the fish of the low dose group, the intercellular space between liver cells expanded slightly, and some of the gill pieces were shed. However, in the fish of the high-dose group, the seriously disordered arrangement and vacuolization of liver cells, atrophy of the nucleus, shedding and lamellae fusion of the gill epithelial and gill piece, vascular atrophy and deformation of the gill filament were observed.In order to explore the toxicologicalmechanismof EE₂ onthe liver and gill of female zebrafish, the antioxidant activity of enzymes（SOD, CAT, and GPx） and the content of MDA（lipid oxidation indicator） in liver and gill were determined. In thelow-dose group, hepatic GPx activity decreased significantly after 3-day exposure, but subsequently returned to normal. However, no significant differences in the activities of SOD and CAT, as well as the content of MDA were observed in the liver. In the gill of the low-dose group, SOD activity decreased first and then recovered normally. Furthermore, the GPx activity on day 3 and day 6 significantly reduced, while CAT activity and the MDA level unchanged. These results indicated low-dose exposure to EE₂ did not causesevere oxidative damage in the liver and gill of zebrafish. In the high-dose group, hepatic GPx activity decreased first and then recovered normally. The activities of SOD and CAT decreased prolong exposure time, while the MDA content increased significantly, indicating that oxidative damage of liver induced by EE₂. In the gill, the activities of SOD, CAT and GPx significantly reduced while the content of MDA increased after 3 days and 6 days of exposure, suggesting that gill was also oxidative damaged, which was consisted with histological findings. In addition, Na +/K +- ATPase activity in the gill was measured and the results showed that it was significantlyinhibited in the high-dose group, combining with gill histology, we inferred that EE₂ canaffected the osmotic pressure regulation, resulting in abdominal dropsy of female zebrafish.