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Role of inhbaa in the partial rescue of vitellogenic growth in bmp15-/- by inha-/-. (A) Morphology and histology of the gonads in different genotypes at 120 dpf. The asterisk shows vitellogenic follicles without yolk mass in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). (B) Oocytes from diffident genotypes at higher magnification. The oocytes in bmp15-/- single mutant were arrested at PV stage with cortical alveoli (CA) but no yolk granules (YG). The double mutant with inha-/- showed normal vitellogenic growth beyond PV stage with both CA and YG, while the lack of inhbaa in the triple mutant blocked YG accumulation again in the oocytes. The double arrowed bar shows the region of clear cytosol between the germinal vesicle (GV) and CA zone where the YG is supposed to be located. (C) Follicle distribution in the ovary of diffident genotypes. The follicles could grow to the size range of PV (~250 μm), MV (~450 μm) and EV stage (~350 μm) in the single (bmp15-/-), double (bmp15-/-;inha-/-) and triple mutant (bmp15-/-;inha-/-;inhbaa-/-), respectively. (D) GSI of females in different genotypes at 120 dpf. The values are expressed as mean ± SEM (n ≥ 5) and analyzed by ANOVA followed by the Tukey HSD for multiple comparisons. Different letters indicate statistical significance (P < 0.05). (E and F) Serum levels of E2 and Vtg in different genotypes at 120 dpf (n = 5). The normal concentrations of E2 and Vtg in the double mutants decreased again by the loss of inhbaa in the triple mutants. The values are expressed as mean ± SEM and analyzed by ANOVA followed by the Tukey HSD for multiple comparisons. Different letters indicate statistical significance (P < 0.05). (G) Fertility test of different genotypes at 120 dpf. Five female fish from each type were tested 10 times with normal WT males by natural breeding. The number of fertilized eggs was counted and analyzed after each breeding. The inha mutant females were sub-fertile with much lower fecundity than the control, while other genotypes of the mutant females were all infertile. (H) Sex ratios of different genotypes at 120 dpf. The double mutant and triple mutant females could maintain sexuality with sex ratio being around 50:50 (♂: ♀), whereas sex reversal occurred in bmp15-/- fish. I-IV, stages of sex reversal; *** P < 0.001 by Chi-squared test compared with the control.
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