Fig. S1

Effects of different temperatures on zebrafish spermatogenesis. A. More advanced labeled germ cell after 2 h, 12 h, 1d, 2d, 3d, 4d, 5d, 6d of 3H-thymidine injection in zebrafish males kept at 20°C, 27°C, 30°C, and 35°C. Leptotene/zygotene (L/Z) and pachytene (P) spermatocytes, metaphase I (MI), initial spermatids (E1), intermediate spermatids (E2), final spermatids (E3) and spermatozoa (Z). Scale bars = 10 μm. Cells were considered labeled when four to five or more grains were present over the nucleus in the presence of low-to-moderate background (i.e., very few grains per histological field observed under oil immersion). Black squares indicate the time in which labeled spermatozoa were found in the lumen of zebrafish testes at different temperatures. B. Histogram showing the combined duration of meiotic and spermiogenic phases at different temperatures. Spermatogenesis did not progress beyond the first meiotic division at 35°C. X axis represents the time, whereas y axis the 3H-thymidine labeled germ cell [Leptotene/zygotene (L/Z), pachytene (P) and diplotene (D) spermatocytes, metaphase I (MI), initial spermatids (E1), intermediate spermatids (E2), final spermatids (E3) and spermatozoa (Z)]. C–E. Histological sections of zebrafish testes at 27°C, 30°C, and 35°C. Sperm free and a massive germ cell apoptosis is seen in zebrafish seminiferous tubules at 35°C. Scale bars = 50 μm.