Testicular toxicity of methyl thiophanate in the Italian wall lizard (Podarcis sicula): morphological and molecular evaluation.

The effects of the fungicide methyl thiophanate (MT) on testis were determined in the Italian wall lizard (Podarcis sicula) using morphological and molecular analyzes. Three experimental trials were performed: an acute test using six doses, a two-week chronic test, and "ecotoxicological" exposure (3 weeks). The minimal lethal dose (LD(50)) of pure MT, reached by the acute test, was 100 mg/kg body weight. Testicular histopathology of surviving animals showed a reduced lumen and several multinucleated giant cells 24 h after injection followed by large decreases in spermatogonia (72%) and secondary spermatocytes (58%) and a loss of spermatids and sperms 7 days after. In the chronic test, a dose equivalent to 1/100 of LD(50) was injected on alternate days. Complete shutting of the lumen and a great decrease in spermatogonia (82%) were observed. In "ecotoxicological" exposure, achieved with a commercial MT compound, testis showed a decrease in primary spermatocytes (20%) and several vacuoles. An increase in germ cell apoptosis was observed in all experimental groups using TUNEL assay. A decrease in expression of androgen and estrogen receptor (AR and ER) mRNAs was seen in all experimental groups. The reduction in AR and ER mRNAs was correlated to exposure time. Indeed, in the "ecotoxicological" treatment (30 days), the decrease reached 82 and 90% for AR and ER mRNAs, respectively. These data strongly indicate that treatment with MT, damaging the seminiferous epithelium and decreasing steroid receptor expression, might render exposed lizards infertile.