Effects of methotrexate exposure on medaka (Oryzias latipes) testes

Abstract

For the purpose of clarifying the histopathological effects of methotrexate (MTX) on medaka testes, wild-type and homogenic p53-deficient male medaka at 4 to 6 months post-hatching were exposed to 0.25 mg/ml of MTX for 96 hr with histopathological examination of testes at 24, 48, 72 and 96 hr. At 72 and 96 hr after the start of MTX exposure, numerous apoptotic cells were observed in the spermatogonia and spermatocytes, and the pyknotic cell rate and the TUNEL-positive and cleaved caspase-3-positive rates in the spermatogonia and spermatocytes of MTX-treated wild type medaka were higher compared with those in the control wild-type medaka. Starting at 48 hr, the phospho-histone H3-positive rate in the spermatogonia and spermatocytes of was significantly lower in MTX-treated wild-type medaka than in control wild-type medaka. In homogenic p53-deficient medaka, apoptosis was not induced in the spermatogonia and spermatocytes by exposure to MTX. Starting at 48 hr, the phospho-histone H3-positive rate in spermatogonia and spermatocytes of MTX-treated homogenic p53-deficient medaka was lower than in control homogenic p53-deficient medaka. Throughout the entire experimental period, there were no significant differences in phospho-histone H3-positive rates in the spermatogonia and spermatocytes between the MTX-treated homogenic p53-deficient medaka group and the MTX-treated wild-type medaka group. In the present study, spermatogonia and spermatocytes of medaka testes were sensitive to MTX at 0.25 mg/ml in the culture water, and MTX-induced apoptosis in the testes was dependent on p53 expression; however, inhibition of MTX-induced cell proliferation was independent of p53 expression.