Roles Of Gsdf And Wt1 In Sex Differentiation And Gonadal Development In Tilapia

Many aquaculture species displayed obvious sexual dimorphic growth rates. Application of sex control technologies in fish breeding has provided significant approaches to increase the production and efficiency in aquaculture. Sex dimorphism favoring males and unwanted early reproduction are the two factors that have made all-male tilapia farming the industry standard. Nile tilapia, Oreochromis niloticus, is a worldwide aquaculture species. It has a predominantly monofactorial mechanism of sex determination with an XX/XY sex determination system. Base on this, genetic male tilapia(GMT) technology has been proposed, i.e., all male XY progenies are produced by mating YY supermales with the normal XX female fish. However, up to now GMT technology has not been applied widely to aquacultural practice due to the difficulty in establisment of YY strain. Induction of sex reversal of YY fish is very difficult, which requies higher hormone concentration and longer treatment period. Further more, the fecundity of the YY female is extremely low. The induced YY female in our laboratory is often infertile. It is urgent for us to study the sex determination and differentiation mechanism in Nile tilapia to provide theory and techniques for the final realization of the genetic sex control. gsdf is a fish specific TGF-β superfamily member with dominant expression in the testis. Recent studies in medaka suggest it is important for testicular differentiation. Wt1 is reported to be involved in the male sex determination and differentiation in mammals. However, its role in fish remains to be elucidated. Teleost fish possess two wt1 paralogs, wt1 a and wt1 b, due to the fish specific genome duplication(3R). In the present study, gsdf and wt1 knockout lines were established using CRISPR/Cas9 in Nile tilapia. The roles of gsdf and wt1 in sex differentiation and gonadal development were studied based on these knockout lines. The gsdf was also mutated in the hybrid ZX tilapia(Nile tilapia XX ♀ × blue tilapia ZZ ♂) to confirm whether the function of Gsdf in sex differentiation in fish is conserved or not. The main results are as follows:1. Positive F0 gsdf mutants induced by CRISPR/Cas9 were obtained in our group previously. The gsdf homozygous knockout line was established using the F0 mutants in the present study. The relationship between mutation rate and gonadal phenotype of the F0 XY mutants, and the phenotype of the gsdf F1 heterozygous and F2 homozygous mutants were analyzed. F0 gsdf-deficient XY fish with high mutation rate(≥58%) developed as intersex, with ovotestes at 90 days after hatching(dah), and become completely sex-reversed with ovaries at 180 and 240 dah. Those F0 individuals with a low mutation rate(<58%) and F1 XY gsdf +/- fish developed as males with normal testes, while the F2 XY gsdf-/- fish developed as females with normal ovaries. The YYgsdf-/- fish were obtained by mated the F1 XYgsdf +/- male fish with the sex reversed F0 XY female fish. The YY gsdf-/- fish developed as female with ovaries at 60 dah by histological observation. These results demonstrated that gsdf is essential for testicular differentiation in Nile tilapia. A threshold level of gsdf expression is required for maleness in tilapia.2. The gene expression in gsdf homozygous mutation gonads was analysed by IHC(immunohistochemistry). The XY gsdf-/- gonads first expressed Dmrt1 which initiated the male pathway at 10 dah, and then activated both male and female pathways as reflected by the expression of Dmrt1 and Cyp19a1 a simultaneously at 18 dah. By 36 dah the XY gsdf-/- gonads have shifted to the female pathway expressing Cyp19a1 a only and developed phase I oocytes indicating sex reversal into ovaries. The male pathway and Dmrt1 expression was initiated, but failed to be maintained in the absence of Gsdf. IHC analyses of the adjacent sections in series from normal XY gonads revealed that Gsdf and Dmrt1 were not expressed at 4 dah, but were both enriched in Sertoli cells in the testis from 5 dah. In silco analysis revealed a Dmrt1 binding site in the promoter of gsdf. Luciferase assay using HEK293 cells revealed that Dmrt1 alone could not activate gsdf gene transcription, however, when co-transfected with Sf1, Dmrt1 enhanced gsdf expression in a dose-dependent manner. Taken together, we conclude that gsdf is a downstream gene of dmrt1 that functions in the male pathway of the Nile tilapia.3. The gene expression of the XY gsdf-/- gonads was analyzed by real-time PCR at 90 dah. The abundance of foxl2, which could up-regulate the expression of cyp19a1 a, was significantly elevated; while the abundance of cyp11b2 was significantly down-regulated in the XY gsdf-/- gonads compared to that of the XY gsdf +/+ gonads, but both were not different from those of the XX gsdf +/+ and gsdf-/- gonads. Consistantly, serum E2(estradiol-17β) level was up-regluated, while 11-KT(11-ketotestosterons) level was down-regulated in the XY gsdf-/- fish compared with that of XY gsdf +/+ fish. AI(aromatase inhibitor) treatment from 10 to 35 dah rescued the XY gsdf-/- gonads into normal testes expressing Dmrt1 and Cyp11b2. The rescued XY gsdf-/- fish produced fertile sperms at 180 dah. These results indicated that Gsdf promotes testicular differentiation by down-regulation of E2 and up-regulation of 11-KT synethsis. However, the involved mechanism remains to be elucidated.4. We introduced blue tilapia and established pure and hybrid lines. When blue tilapia ZZ male crossed with Nile tilapia XX female, 100% male hybrid F1 ZX progeny were obtained. When the hybrid F1 ZX male backcrossed with the Nile tilapia XX female, sex ratio of the progeny was 1:1. When blue tilapia ZW female crossed with Nile tilapia YY super-male, 100% male hybrid F1 progeny was obtained. When ZZ male crossed with ZW female within blue tilapia, sex ratio of the progeny was 1:1. The aromatase inhibitor Fadrozole treatment could reverse the sex of the ZW fish. When blue tilapia ZW male crossed with Nile tilapia XX female, sex ratio of the progeny was 1:1. When crossed the ZW male and ZW female within blue tilapia, sex ratio of the progeny was 1:3. Combined with the fact that the XX is female and XY is male in Nile tilapia, while ZZ is male and ZW is female in blue tilapia, we concluded that the order of sex determination is Y>W>Z>X in tilapia. The blue tilapia gsdf ORF sequences were obtained from its gonadal transcriptome data. The similarity between Nile tilapia and blue tilapia gsdf ORF is 99.5%. The Gsdf exepression in the ZX hybrid gonad was analysed by IHC, Gsdf was found to be predominantly expressed in the ZX hybrid gonad as in the XY gonad at 10 dah. Knockout of gsdf was performed using CRISPR/Cas9 in the ZX hybrid, nearly half of the F0 mutants displayed male to female sex reversal. These results indicated that Gsdf has conserved functions in testicular differentiation at different genetic background.5. The expression patterns of wt1 a and wt1 b were analysed by in situ hybridization. In the kidney, wt1 a was expressed in the glomerulus at 3, 5, 20 and 40 dah, while wt1 b was expressed in the glomerulus at 5, 20 and 40 dah. In the gonad, both wt1 a and wt1 b were expressed in the somatic cells in both sexes at 3 and 5 dah, and wt1 a in the granulose cells, thecal cells and interstial cells of the ovary and Sertoli cells of the testis, while wt1 b in the interstitial cells of the ovary and Leydig cell of the testis at adult stages. We successfully mutated by CRISPR/Cas9 and established wt1 a and wt1 b knockout lines in Nile tilapia. The F2 wt1a-/- fish were with serious edema at 6 dah, and all of them were died at 10 dah. wt1b-/- mutation was not lethal. Histological observations revealed that the glomerulus in the kidney of wt1a-/- fish were not formed, while the they were normal in the kidney of wt1b-/- and wild type fish at 6 dah. These results indicated that wt1 a is critical for the glomerular formation which could not be compensated by wt1 b. Histological observation and IHC analyses at 180 dah revealed that the XX wt1b-/- gonads developed as normal ovaries, expressing Cyp19a1 a but not expressing Cyp11b2, and XY wt1b-/- gonads developed as normal testes, expressing Cyp11b2 but not expressing Cyp19a1 a. Consistently, the serum E2 and 11-KT levels displayed no differences between the XX wt1b-/- mutants and the XX control(wt1b+/+ and wt1b+/-) fish, and the serum E2 and 11-KT levels displayed no differences between the XY wt1b-/- mutants and the XY control(wt1b+/+ and wt1b+/-) fish. The XY wt1b-/-male produced fertile sperms at 180 dah. The wt1 a homozygous mutation is lethal, and therefore, the function of wt1 a in sex determination can not be judged. The wt1 b homozygous mutation is not lethal and can not influence the sex determination. The function of wt1 b in testis development might be redundant, while its function in ovary development remains to be enucidated.In summary, the homozygous mutation of gsdf in XY and YY fish resulted in male to female sex reversal, indicating its essential role in testicular differentiation and providing a new approach for the establishment of YY strain in tilapia. The expression of Dmrt1 in the gsdf-/- XY gonand and the ability of Dmrt1 in binding to the promoter and activation of gsdf transcription in the presence of Sf1 suggested gsdf is downstream gene of dmrt1 in male pathway of Nile tilapia. Gsdf promotes male sex differentiation via down-regulaiton of the E2 and up-regulaiton of the 11-KT synethsis. As in XY fish, mutation of gsdf in ZX fish also resulted in male to female sex reversal, indicating that the function of Gsdf in testicular differneation is conserved in tilapia with different genetic background. The wt1 a is vital for survival of tilapia as its homozygous mutation is lethal, which could not be compensated by wt1 b. wt1 b is redundant in tilapia gonad as its homozygous mutaions resulted in no clear phenotype in terms of sex differentiation and gonadal development.The function of wt1 a in sex determination can not be judged as homozygous mutation is lethal, wt1 b homozygous mutation did not influence the sex determination. At adult stage, wt1 b homozygous mutations can not influnce the male reproduction, while it is unclear about infulences of wt1 b homozygous mutationits on female reproduction.