Molecular Cloning, Expression And Characterization Of Several Genes (Rspo1, β-catenin And TSP-1) Involved In Ovarian Differentiation And Development In Tilapia

Recently, there are many reports light on the molecular mechanisms of mammalian female sex determination and suggest that the Wnt4/Rspol/β-catenin pathway and Foxl2 act in a complementary manner to determine ovarian differntiation and to antagonize testicular development, while P-catenin is a key protein in antagonism between the male and female pathways. In contrast, sex determination and differentiation of non-mammalian vertebrates are controlled by both genetic and environmental factors, e.g. hormones, especially estrogen which is a natural inducer for ovarian differentiation in non-mammalian vertebrates. However, studies on the roles of Wnt4/Rspol/p-catenin signaling pathway in sex determination and differentiation were mainly focused on mammals. There is no such report on non-mammalian vertebrates. In the present study, Rspol, twoβ-catenin (named asβ-catenin-1 and-2, abbreviated asβ-cat-1 and-2) encoded by two different genes were identified and cloned from the Nile tilapia (Oreochromis niloticus) ovary by RT-PCR and subsequent RACE. Phylogenetic analysis of P-catenin from various vertebrates indicated that the co-existence of twoβ-catenins is an unique phenomenon in teleosts and are probably derived from the additional genome duplication (the fish-specific genome duplication, FGSD) that occurred at the emergence of modern fish. The tissue distribution analysis by RT-PCR revealed that tilapia Rspol was ubiquitously expressed in all examined tissues,β-cat-1 and-2 were expressed in brain, pituitary, gill, heart, kidney, intestine and gonads. In the gonads, the expression levels of Rspol and P-cat-2 were higher in the ovary than in the testis, whereasβ-cat-1 was exclusively expressed in the ovary. Moreover, the gonadal cell types where Rspol and two types ofβ-cat express were further analyzed by in situ hybridization. Both of them were expressed in oocytes at phaseⅠ,ⅡandⅢof ovary, not in somatic cells, while Rspol was also expressed in spermatogonium and spermatocytes. It is worth noting that the expression level ofβ-cat gradually increases with the ovarain development from 10 days after hatching to latter stage in primary oocytes at phaseⅠ,ⅡandⅢImmunochemistry ananlyses further confirmed that tilapiaβ-catenin was localized in the germ cells of ovary, suggesting that P-catenin might be an important factor involved in teleost ovary differentiation and development. Furthermore, Luciferase assays were performed to study the effect of P-catenin on the Cyp19ala gene transcriptional activity and the relationship between P-catenin and the known pathway of estrogen synthesis which was controlled by Foxl2 and Dmrtl. The results showed thatβ-cat-1 and-2 alone was unable to activate Cyp19ala transcription, however, both of them enhanced Sfl-mediated Cypl9ala transcription in HEK293 cells. Moreover, Foxl2 and P-cat-2 synergistically enhanced the Sfl-mediated Cypl9ala gene transcription, while Dmrtl suppressed the P-cat-2 enhanced and Sfl activated Cypl9ala promoter activity. The results indicate that both Rspol/β-catenin-mediated germ cell pathway and Foxl2/Dmrtl-mediated somatic cell pathway might control the production of estrogen in the gonad through transcriptional regulation of aromatase gene, which in turn, determine the gonadal differentiation and maintain the gonadal function.Estrogen and factors within the extracellular matrix (ECM) have been reported to. be important in the vertebrate follicular development and ovarian cycle. Thromobospondins (TSPs 1-5) are important members of the ECM proteins. In order to address the roles of TSP-1 in the teleost gonadal development and spawning cycle, two types of TSP-1 (named TSP-1a and TSP-1b) were cloned from Nile tilapia. Phylogenetic analysis of these TSP-1 sequences, together with those available from other vertebrates further demonstrated that two types of TSP-1 exist only in teleosts, extending the finding in fugu and tetraodon to two additional fish species. The expression of both genes was examined using tilapia at various developmental stages. Tilapia TSP-1a and TSP-1b were each expressed in a wide range of tissues examined. The early expression of TSP-1b in both XX and XY gonads from 5 dah onwards suggested an important role in the formation of gonads, while the expression of TSP-1a only in ovaries during later stages of development (from 120 dah onwards) may suggest that TSP-1a is involved in vitellogenesis. In situ hybridization analyses revealed differential expression in adult fish, with TSP-1a occurring in granulosa cells and TSP-1b in theca cells and in the epithelial cells of the efferent duct of the testis. During the 14-day spawning cycle, the expression of both types of TSP-1 exhibited distinct peaks at day 5 (peak of vitellogenesis) and day 12 (oocyte maturation), corresponding to active estrogen production and vitellogenesis and to the synthesis of FSH, LH and oocyte maturation, suggesting that they have important roles in spawning cycle of the Nile tilapia.Fish gonad differentiation and development are mediated by local factors, such as a variety of hormones and growth factors, produced in an autocrine and paracrine fashion by gonad somatic cell and germ cell. They form an intimate regulatory network and interact with each other. In present study, three gonadotropin subunit cDNAs, GtHa, FSHfβand LHβ, were isolated from Southern catfish (Silurus meridionalis Chen) ovary. RT-PCR revealed that GtHa, FSHβand LHβmRNA were expressed in ovary, female and male pituitaries, but not in testis. Ontogenic study showed that GtHa and FSHβexpressed in ovary from 25 dah and LHβexpressed from 40dah onwards. These results indicated the involvement of the local gonadotropin in the proliferation of Southern catfish primordial germ cell and early ovrian development. All-female Southern catfish fry were treated with estrogen receptor tamoxifen (TAM) from 5 to 25dah). The expressions of the three subunits were down-regulated but not vanished in TAM treated gonad as compared with control. Some partial and complete sex reversed fish was observed in TAM group. Together with other findings reported in other fish, it is conceivable for us to presume that the secretion of gonadotropin in both pituitary and gonads might have important roles on sex steroids synthesis and interacted with those factors directly or indirectly in undifferentiated gonad and in turn on sex differentiation through brain-pituitary-gonad axis.In summary, Rspol, two types ofβ-catenin and TSP-1 were cloned from tilapia. Tissue distribution, cell-specific expressions and expression profile during the spawning cycle were studied by RT-PCR, real time PCR, in situ hybridization and immunochemistry. Moreover, effects of tilapiaβ-catenin on Cypl9ala promoter activity were further investigated by luciferase assay. Our results indicate that these genes might have important roles in fish ovarian differentiation and development. These findings highlight the relationship between theβ-catenin-mediated germ cell pathway and the known Foxl2/Dmrtl-mediated somatic cell pathway in the control of estrogen synthesis and sex differentiation; provide basic data for the research on the molecular mechanism of ovarian differentiation and development, and the crosstalk between germ cell and somatic cell in fish.