Arsenic inhibits in vitro spermatogenesis and induces germ cell apoptosis in Japanese eel (Anguilla japonica)

Abstract

The precise mechanism and direct effects of arsenic on fish, particularly in reproduction, are not well clarified. The aim of this study is to investigate the direct influence of arsenic on fish spermatogenesis using the Japanese eel (Anguilla japonica) in vitro testicular organ culture system. Eel testicular fragments were cultured in vitro with 0.1-100 microM arsenic with or without human chorionic gonadotropin (hCG) for 6 or 15 days at 20 degrees C. Arsenic treatment provoked a dose-dependent inhibition of hCG-induced germ cell proliferation as revealed by 5-bromo-2-deoxyuridine immunohistochemistry. Time-resolved fluorescent immunoassay showed that arsenic suppressed hCG-induced synthesis of 11-ketotestosterone (11-KT) in testicular fragments incubated with 0.0001-100 microM arsenic and hCG for 18 h. A 0.1 microM (7 microg/l) dose of arsenic which is lower than the World Health Organization drinking water quality guideline of 10 microg/l most effectively reduced 11-KT production. The hCG-induced synthesis of progesterone from pregnenolone was significantly inhibited by low doses of arsenic (0.1-1 microM), implying an inhibition of 3beta-hydroxysteroid dehydrogenase activity. In situ TUNEL assays indicated that germ cells undergo apoptosis at the highest dose of arsenic (100 microM). An arsenic concentration-dependent increase in oxidative DNA damage was detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunohistochemistry. A peak in 8-OHdG index was observed in testicular fragments treated with 100 microM arsenic and hCG consistent with the TUNEL results. These data suggest that low doses of arsenic may inhibit spermatogenesis via steroidogenesis suppression, while high doses of arsenic induce oxidative stress-mediated germ cell apoptosis.