Fig 1

(A) Generation of gdf9 mutant by TALEN. The target site on chromosome 14 for left and right TALE proteins are boxed. A mutant with 5-bp deletion was obtained with a premature stop codon (TGA), which generated a truncated protein with 32 amino acids (15 from the Gdf9 precursor). RT-PCR analysis on mRNA from the ovary using a WT-specific primer (F) showed a positive signal in the control ovary (gdf9+/-), but not in the mutant (gdf9-/-). (B) Sex ratios during and after gonadal differentiation in gdf9 mutant fish. The offspring from one crossing (gdf9+/- female X gdf9-/- male) were sampled at different times from 35 to 130 dpf for genotyping and sex identification by histology. The sex ratios in the post-differentiation period from 35 to 55 dpf remained relatively constant and comparable to those in the control fish. However, the ratio changed gradually but significantly afterwards towards all males. The number of fish examined is shown at the bottom of each column. *** P < 0.001 (X² test). (C) The ovary and testis in the control (gdf9+/-) and mutant (gdf9-/-) at 90 dpf. The control females had reached sexual maturity with all stages of follicles present in the ovary (PG, primary growth; PV, pre-vitellogenic; MV, mid-vitellogenic; LV, late vitellogenic; and FG, full-grown) while the mutant ovary contained PG follicles only. The testis development and spermatogenesis showed no difference between mutant and control males. SG, spermatogonia; SC, spermatocytes; SZ, spermatozoa. The number in each photo indicates the number of fish examined with the same phenotype.