We describe here the isolation and cytogenetic characterization of three meiotic prophase I mutants, denoted ietsugu (its), iesada (isa), and iemochi (imo), isolated by a novel N-ethyl-N-nitrosourea mutagenesis screen for adult zebrafish gonadogenesis. Histological examination and flow cytometry analysis of testes from these mutants showed that each contained neither spermatids nor sperm. Staining for Sycp3 and cleaved Caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) assay further revealed that its had defects at the onset of meiosis, and that isa and imo spermatocytes failed to progress past the zygotene stage with apoptosis occurring in the testicular somatic cells. Staining for phosphorylated histone H2AX showed that foci formation in leptotene spermatocytes was disrupted in isa and imo. Furthermore, in vitro differentiation experiments revealed the possibility that the defects and sterility associated with mutations were germ line autonomous. Our results thus indicate that each responsible gene is necessary for meiotic progression during spermatogenesis and for male fertility.
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