In teleost fish, sex steroids are involved in sex determination, sex differentiation, and fertility. Cyp17a1 (Cytochrome P450 family 17 subfamily A member 1) is thought to play essential roles in fish steroidogenesis. Therefore, to further understand its roles in steroidogenesis, sex determination, and fertility in fish, we constructed a cyp17a1 gene mutant in Nile tilapia (Oreochromis niloticus). In XX fish, mutation of the cyp17a1 gene led to a female-to-male sex reversal with a significant decline in 17β-estradiol (E2) and testosterone (T) production, and ectopic expression of male-biased markers (Dmrt1 and Gsdf) in gonads from the critical window of sex determination. Sex reversal was successfully rescued via T or E2 administration, and ovarian characteristics were maintained after termination of E2 supplementation in the absence of endogenous estrogen production in cyp17a1^–/– XX fish. Likewise, deficiencies in T and 11-ketotestosterone (11-KT) production in both cyp17a1^–/– XX sex-reversed males and cyp17a1^–/– XY mutants resulted in meiotic initiation delays, vas deferens obstruction and sterility due to excessive apoptosis and abnormal mitochondrial morphology. However, 11-KT treatment successfully rescued the dysspermia to produce normal sperm in cyp17a1^–/– male fish. Significant increases in gonadotropic hormone (gth) and gth receptors in cyp17a1^–/– mutants may excessively upregulate steroidogenic gene expression in Leydig cells through a feedback loop. Taken together, our findings demonstrate that Cyp17a1 is indispensable for E2 production, which is fundamental for female sex determination and differentiation in XX tilapia. Additionally, Cyp17a1 is essential for T and 11-KT production, which further promotes spermatogenesis and fertility in XY males.

Background: In mammals, R-spondin (Rspo), an activator of the Wnt/β-catenin signaling pathway, has been shown to be involved in ovarian differentiation. However, the role of the Rspo/Wnt/β-catenin signaling pathway in fish gonads is still unknown. Results: In the present study, full-length cDNAs of Rspo1, 2 and 3 were cloned from the gonads of medaka (Oryzias latipes). The deduced amino acid sequences of mRspo1-3 were shown to have a similar structural organization. Phylogenetic analysis showed that Rspo1, 2 and 3 were specifically clustered into three distinct clads. Tissue distribution revealed that three Rspo genes were abundantly expressed in the brain and ovary. Real-time PCR analysis around hatching (S33-5dah) demonstrated that three Rspo genes were specifically enhanced in female gonads from S38. In situ hybridization (ISH) analysis demonstrated that three Rspo genes were expressed in the germ cell in ovary, but not in testis. Fluorescence multi-color ISH showed that Rspo1 was expressed in both somatic cells and germ cells at 10dah. Exposure to ethinylestradiol (EE2) in XY individuals for one week dramatically enhanced the expression of three Rspo genes both at 0dah and in adulthood. Conclusions: These results suggest that the Rspo-activating signaling pathway is involved in the ovarian differentiation and maintenance in medaka.

Figla (factor in the germ line, alpha), a female germ cell-specific transcription factor, had been shown to activate genetic hierarchies in oocytes. The ectopic expression of Figla was known to repress spermatogenesis-associated genes in male mice. However, the potential role of Figla in other vertebrates remains elusive. The present work was aimed to identify and characterize the functional relevance of Figla in the ovarian development of Nile tilapia (Oreochromis niloticus). Tissue distribution and ontogeny analysis revealed that tilapia Figla gene was dominantly expressed in the ovary from 30 days after hatching. Immunohistochemistry analysis also demonstrated that Figla was expressed in the cytoplasm of early primary oocytes. Intriguingly, over-expression of Figla in XY fish resulted in the disruption of spermatogenesis along with the depletion of meiotic spermatocytes and spermatids in testis. Dramatic decline of sycp3 (synaptonemal complex protein 3) and prm (protamine) expression indicates that meiotic spermatocytes and mature sperm production are impaired. Even though Sertoli cell (dmrt1) and Leydig cell (star and cyp17a1) marker genes remained unaffected, hsd3b1 expression and 11-KT production were enhanced in Figla-transgene testis. Taken together, our data suggest that fish Figla might play an essential role in the ovarian development by antagonizing spermatogenesis.