Courtship parameters of wild-type male, tdrd12^−/−, cyp17a1.^−/− and double knockout (KO) fish with wild-type females. Twelve individuals of each genotype were selected and randomly paired with wild-type females, and all pairs were exposed to light for 20 min before the mating partition was removed. A Frequency of intimate contact with wild-type females during the courtship period. B Duration of intimate contact time with females during the courtship period. C Average frequency of intimate contact with females per minute during the courtship period. D Average duration of intimate contact time with females per minute during the courtship period. E Representative mating behaviors of males of four genotypes with wild-type females. *Significant difference (P < 0.05); **significant difference (P < 0.01); ***significant difference P < 0.001

Identification of secondary sex characteristics (SSCs). A–D SSCs of wild-type females: body shape/body color (A), anal fin and anal papilla (B), caudal fin (C) and pectoral fin (D) without breeding tubercles. E–H SSCs of wild-type males: body shape/color (E), anal fin (F), caudal fin (G) and pectoral fin (H). The arrow indicates a large number of breeding tubercles, which occur specifically in males. H–K SSCs of cyp17a1^+/+; tdrd12^−/−: body shape/body color (H), anal fin (I), caudal fin (J) and pectoral fin (K) with breeding tubercles. L–O SSCs of cyp17a1^−/−: body shape/body color (L), anal fin (M), caudal fin (N) and pectoral fin (O) without breeding tubercles. P–S SSCs of double KO mutants: body shape/body color (P), anal fin (Q), caudal fin (R) and pectoral fin (S) without breeding tubercles. mpf: months post-fertilization; PF: pectoral fin; AF: anal fin; CF: caudal fin. 6 individuals of each genotype were detected in this assay

Morphology of primary sex characteristics (PSCs) of the mutants. A–A″) Normal testis of a control male. Anatomical view (A); histological view of the whole testis (A′, testis outlined in white); enlarged view shows a well-developed testis with various developmental stages of germ cells, such as spermatocytes (SC), spermatogonia (SG), sperm (SP) and somatic cells (ST) in the testis (A″). B–B″ Atrophic testis of a tdrd12^−/− fish; the testis lacks germ cells and is surrounded by fatty layers. Anatomical view (B), histological view (B′, outlined in white) and enlarged view (B″). C–C″ Testis of a cyp17a1^−/− fish. Anatomical view; C histological view (C′, testis outlined in white); enlarged view shows the testis with various developmental stages of germ cells, such as SC, SG, sperm and some ST (C″). D–D″ Atrophic testis of a double knockout (KO) fish. Anatomical view (D); histological view (D′, testis outlined in white); enlarged view (D″) shows an atrophic testis similar to that of the tdrd12^−/− fish. 6 fish of each genotype were used in this assay

Hormone concentrations of testosterone (T), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) in the gonads, serum, and brain of control males, tdrd12^−/− fish, cyp17a1.^−/− fish, double knockout (KO) fish and control females. A–C T levels in the gonads, blood, and brain samples. D–F 11-KT levels in the gonads, blood, and brain samples. C E2 levels in the gonads, blood, and brain samples. For this assay, the numbers of gonadal, blood, and brain samples were n = 4, n = 6 and n = 8; Error bars represent the mean ± SEM. Bars with different letters are significantly different from each other, P < 0.05

RT‒qPCR was performed to validate the RNA transcriptome data in different brain region samples. A–D nr2e3, dio2, cyp19a1b, and igf1 in the forebrain; E–H egr2b, gh1, slc25a18, klf3, and dio2 in the midbrain; I–L sox19a, ddx43, npas2, klf3, and shisa19 in the hindbrain. Error bars represent the mean ± SD; bars with different letters are significantly different from each other, P < 0.05. Brain samples were n = 5 in biological triplicates

Venn diagram for significantly differentially expressed genes (DEGs) in tdrd12^−/− fish, cyp17a1^−/− fish, double knockout (KO), and wild-type female fish compared with wild-type males in the forebrain (A, B), midbrain (C, D), and hindbrain (E, F) (P < 0.05, |fold change|> 2). The drop-shaped regions with thick black outlines shows those shared genes are shown in in cyp17a1^−/− and double KO fish compared with wild-type male brains; grey gridline areas shows those co-DEGs shared by cyp17a1^−/−, double KO fish, and females compared with wild-type male brains

Heatmap shows the top Gene Ontology (GO) terms of male-biased genes, differentially expressed genes (DEGs) in cyp17a1.^−/− fish, and DEGs in double knockout (KO) fish in comparison with wild-type males in the forebrain (A), midbrain (B) and hindbrain (C)

Heat map shows the representative co-downregulated (upper) and co-upregulated (down) gene expression in wild-type females, tdrd12^−/− fish, cyp17a1.^−/− fish or double KO fish when compared with wild-type males in the forebrain (A), midbrain (B) and hindbrain (C)