Roles Of Rspo1,β-catenin In Ovarian Differentiation And Maintenance In Nile Tilapia

For a long time, sex determination and differentiation has been a concern in the field of reproductive biology. Early ovarian development has long been thought of as a default pathway switched on passively by the absence of Sry gene. Recently, there are many reports demonstrate that the molecular mechanisms of mammalian female sex determination is a strict molecular cascade and involves many transcription factors and signal pathways. The sex determination in the mammalian bipotential gonads is controlled by an antagonistic process between the male(Sry/Sox9/Fgf9) and female(Rspo1/Wnt/β-catenin and Foxl2) signaling pathways. Expression data indicate that Rspo1/Wnt/β-catenin signaling might also play a role in fish gonadal differentiation. In order to study the roles of Rspo1/Wnt/β-catenin signal pathway in teleosts female sex differentiation, we identified tilapia Rspo1 and two β-catenin genes(β-cat1 and β-cat2), and analyzed their expression pattern in the early female sex differentiation stages. We also investigated their functions in fish ovarian development by knocking down Rspo1, β-cat1 and β-cat2 using the transcription activator-like effector nucleases(TALENs) system. At the same time, we produced the recombinant Dkk1, an inhibitor of the canonical Rspo1/Wnt/β-catenin pathway, and used the recombinant Dkk1 and FH535(a chemical inhibitor of Wnt/β-catenin pathway) to incutate tilapia ovaries in vitro. The main results are as follows: 1. The amino acids of β-cat1 and β-cat2 are conserved at the N-terminal and the central armadillo repeats, but the C-terminal region in β-cat2, comprising part of the activation domain, diverge markedly from those of β-cat1 in fish and β-cat in tetrapod. We generated a fusion protein combining β-cat2 with a strong activation domain VP16(β-cat2-VP16) and a mutant form of β-cat1(β-cat1-C–) in which the last 100 aa of the C-terminal region were deleted. In luciferase assays, β-cat1 alone activated the TOPFlash reporter in a dose-dependent manner, whereas Rspo1 and β-cat2 failed to do so. However, both of them enhanced the β-cat1 mediated TOPFlash reporter activity. Luciferase assay demonstrated that β-cat1-C– caused a marked decline in the activation of the reporter. In contrast, the fusion protein β-cat2-VP16 activated the transcription of the reporter. A specific interaction between β-cat1 and β-cat2 was also observed in the mammalian two-hybrid assay. 2. Real-time PCR analyses showed that Rspo1 exhibit a female-specific increase in the ovaries at 20 dah. Significant increases of β-cat1 and β-cat2 were observed at 20, 60 and 180 dah. 3. Rspo1 deficiency causes the retardation of ovarian development(without oocytes) in XX fish at 60 dah. However, Rspo1 deficiency did not affect the expression of Cyp19a1 a in the somatic cells. At 180 dah, the expression of Cyp11b2 and Dmrt1 increased significantly in Rspo1-deficient ovaries. At the same time, serum 11-KT level was upregulated. The cyp19a1 a level and serum E2 level remained unchanged. 4. Histologically, compared with the control ovaries, there is no meiosis in the β-cat1 or β-cat2 deficient ovaries at 60 dah. Cyp11b2 and Dmrt1(Sertoli cell marker) were ectopically expressed in the β-cat1 or β-cat2 knockdown ovaries at 90 dah. Though ovarian development caught up by 180 dah, the expression of Cyp11b2 was still detected, and serum 11-KT levels were significantly increased in the β-cat1 or β-cat2 deficient but not the control ovaries. In the in vitro cultured 180 dah ovary, treatment with recombinant Dkk1 resulted in significant downregulation of β-cat1 and β-cat2, and upregulation of sox9 and cyp11b2, dmrt1, compared with the control. However, the level of cyp19a1 a remained unchanged. Consistently, IHC analysis also demonstrated that Cyp19a1 a displayed no differences in the Dkk1 treated and control ovaries. Dmrt1 and Cyp11b2, which were not observed in the control ovary, were ectopically expressed in the Dkk1 treated 20 and 180 dah ovaries. 5. In the in vitro cultured 180 dah ovaries, treatment with Dkk1 or 50 μM FH535 for 4 days resulted in significant downregulation of β-cat1 and β-cat2, upregulation of foxl2, cyp11b2, and dmrt1 expression comparing with the control.In conclusion, this study suggests that Rspo1 activated the β-cat1 mediated signal pathway, and the two β-cat genes act synergistically by antagonizing male pathway genes and androgen production during ovarian development. Despite the diversity of sex determinants in different phyla, Rspo1/Wnt/β-catenin signaling appears to be conserved for ovarian differentiation and development in vertebrates.