Developmental expression of the ddx5 gene in zebrafish gonads. The expression of ddx5 was examined by whole-mount in situ hybridization (WISH) using sense ddx5 RNA probes (b, f) and antisense ddx5 RNA probes (a, c–e, g, h). (a, b) 24 hpf embryos. (c) 20 dpf immature gonad. (d, f) 31 dpf thin immature gonads. (e) 31 dpf thick immature gonad. (g) 5 mpf testis. (h) 5 mpf ovary. ddx5 expression was widely detected in 24 hpf embryos (a) and weakly detected in 20 dpf immature gonads (c). The ddx5 gene was strongly expressed in thin and thick immature gonads at 31 dpf (d, e). The ddx5 expression is strongly detected in stage I and II oocytes (h), while the ddx5 gene is weakly expressed in whole testes at 5 mpf (g). Scale bar 200 μm.

Disruption of the ddx5^−/− gene causes abnormal sex ratio. Homozygous ddx5^−/− male mutants (5 mpf) crossed heterozygous ddx5^+/− females (5 mpf), and the sex of growing adult fish was determined by anatomical features (testis or ovary). Total number of progeny: n = 199. Genomic DNA was prepared from individual caudal fins, and the genotype of each fish was determined by genomic PCR using the ddx5 locus-specific primers. The number of heterozygous wild-type animals: n = 112. The number of homozygous mutant animals: n = 87.

Morphology of immature gonads in the ddx5-deficient fish at 31 dpf. (a) Wild-type, thick immature gonad. (b) Wild-type, thin immature gonad. (c) ddx5^−/− mutant, thick immature gonad. (d) ddx5^−/− mutant, thin immature gonad. Scale bar 200 μm.

Dying cells in immature gonads of ddx5-deficient fish. (a–f) TUNEL analysis. (a, b) Wild-type. (c, d) ddx5^−/− mutant. (a, c, e) Thick immature gonads. (b, d, f) Thin immature gonads. Scale bar 200 μm. (e) The number of apoptotic cells in wild-type (e; n = 12, total 94 positive cells) (f; n = 4, total 113 positive cells) and ddx5^−/− mutants (e; n = 7, total 538 positive cells) (f; n = 7, total 160 positive cells) were counted. Error bars indicate standard deviation. Asterisks indicate statistical significance between wild-type and mutant samples (by t test). ***P < 0.001. ns not significant.

Homozygous adult ddx5^−/− females had small ovaries containing immature oocytes. (a, b, e, f) ddx5^+/− wild-type at 5 mpf. (c, d, g, h) The ddx5^−/− mutants at 5 mpf. The ovaries of ddx5-deficient females predominantly possessed stage I and II embryos, while various ovarian stages, from I to IV, were observed in wild-type females. The testes of wild-type and ddx5-deficient fish were similar. Genotyping of individual fish was performed by genomic PCR. (a) Scale bar 5 mm. (b) Scale bar 1 mm.

Distribution of the ddx5-deficient oocytes classified by oocyte maturation. Ovarian follicles from wild-type (n = 5) and mutant animals (n = 5) at 5 mpf were classified by diameter (stage I; 7–140 μm, stage II; 140–340 μm, stage III; 340–690 μm, stage IV, V; 690–750 μm). Fifty ovarian follicles from individual ovaries represent the proportion of oocyte stages. Error bars indicate standard deviation.

Hematoxylin and eosin (HE) staining of gonadal sections. (a, b) ddx5^+/− wild-type gonads. (c, d) the ddx5^−/− mutant gonads. (a, c) Wild-type ovaries possessed stage I–IV oocytes, whereas ddx5-deficient ovaries predominantly contained stage I and II oocytes. (b, d) Both wild-type and ddx5-deficient testes contained differentiating germ cells. Scale bar 200 μm.

cAMP concentration relative to the wild-type ovaries. The concentration of cAMP in each ovary (wild-type: n = 4, ddx5^−/− mutant: n = 4,) was determined with a cAMP ELISA kit. Error bars indicate standard deviation. Asterisks indicate statistical significance between wild-type and the mutant levels (by t test). *P < 0.05.

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