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Updated: Feb 19, 2026
Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes
Published on: October 6, 2022
Effects of methotrexate exposure on medaka (Oryzias latipes) testes
Ayano Hirako1, Yuki Takeoka1, Satoshi Furukawa2
1Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Minami 4-101 Koyama-cho, Tottori,Tottori 680-8553, Japan.
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 h with histopathological examination of testes at 24, 48, 72 and 96 h. At 72 and 96 h 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 h, 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 h, 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.
Insights
Methotrexate (MTX) induces apoptosis in medaka testes, specifically in spermatogonia and spermatocytes. This MTX-induced testicular apoptosis is dependent on p53 expression, but cell proliferation inhibition is not.
Area of Science:
- Reproductive Toxicology
- Molecular Biology
- Histopathology
Background:
- Methotrexate (MTX) is a chemotherapy agent with known toxic effects.
- The impact of MTX on testicular histopathology requires further clarification.
- The role of p53 in MTX-induced testicular toxicity is not fully understood.
Purpose of the Study:
- To investigate the histopathological effects of MTX on medaka testes.
- To determine the role of p53 in MTX-induced apoptosis and cell proliferation in medaka testes.
Main Methods:
- Wild-type and p53-deficient male medaka were exposed to MTX (0.25 mg/ml) for 96 hours.
- Testes were examined histopathologically at 24, 48, 72, and 96 hours.
- Apoptosis was assessed using TUNEL and cleaved caspase-3 staining.
- Cell proliferation was evaluated by phospho-histone H3 staining.
Main Results:
- MTX exposure induced significant apoptosis in spermatogonia and spermatocytes of wild-type medaka.
- MTX-induced apoptosis in testes was absent in p53-deficient medaka.
- MTX significantly reduced cell proliferation in both wild-type and p53-deficient medaka testes.
- Testicular apoptosis induced by MTX was p53-dependent, while proliferation inhibition was p53-independent.
Conclusions:
- Medaka spermatogonia and spermatocytes are sensitive to MTX.
- MTX-induced testicular apoptosis in medaka is mediated by p53.
- Inhibition of cell proliferation by MTX in medaka testes occurs independently of p53.
