Expression Of Gsdf In Gonads And Its Function In Sex Determination Of Tilapia

It is well known that TGF-β superfamily are involved in various physiological process including reproduction, growth and development in vertebrates, gsdf (gonadal soma derived factor) is a new member of TGF-β superfamily. Previous studies had showed that gsdf, mainly expressed in gonads, was only found in teleosts and coelacanthus. Studies on Gsdf were predomintly based on its expression patterns in fish gonads. There were few studies on function of Gsdf in sex determination and differentiation via gain of function and loss of function. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with a stable XX/XY sex determination system, has become one of the most important species in global aquaculture. Moreover, it is a good experimental model for understanding the developmental genetic basis of sex determination because of the availability of monosex populations and the whole genome sequence. In the present study, RT-PCR, real-time PCR and immunohistochemistry were performed to study gsdf/Gsdf expression in tilaipa gonads. In addition, the function of Gsdf in tilaipa sex determination and differentiation was elucidated by transgenic overexpression and knockout. The main results are as follows:1) In the present study, tilapia gsdf gene was isolated from its genome sequences. The sequence analysis showed that gsdf was relatively conserved during the fish evolution. The tilapia gsdf showed the highest similarity to that of zebra mbuna (Maylandia zebra)(90%). The gene structures of gsdf were very conserved among fishes. The syntenic analysis showed gsdf gene localized to a syntenic chromosomal fragment well conserved in fishes. Furthermore, except in teleosts, gsdf was also isolated from elephant shark (Callorhinchus milii), but not from any tetrapods. These findings indicated that gsdf exists not only in teleosts and coelacanthus, but also in chondrichthians. The absence of gsdf in tetrapods might be due to secondary loss.2) Tissue distribution analysis of gsdf by RT-PCR showed that gsdf was found to be mainly expressed in the testis of the adult fish. The expression level of gsdf in the ovary was significantly lower than in the testis. The expression profile of gsdf in gonads during the development was studied by real-time PCR. The expression of gsdf was detected from5dah (days after hatching) in XY gonads, increased from5dah and peaked at30dah, then declined. The expression of gsdf was also observed from5dah in XX gonads, with a similar expression pattern to that in XY gonads. During the development, the expression level of gsdf in XY gonads was significantly higher than that in XX gonads. Furthermore, polyclonal antibody of Gsdf was successfully prepared. By1HC using this antibody, the Gsdf protein expression profile was consistent with the profile obtained from real-time PCR.1HC results also showed that Gsdf was highly expressed in supporting cells surrounding the spermatogonia in the testis from5dah onwards. On the other hand, weak signal was detected in somatic cells surrounding the oogonia from5dah to adult fish. These results suggested that Gsdf might plays a critical role in tilapia male sex determination.3) Disruption of Gsdf in XY tilapia through CRISPR/Cas9system was achieved with high efficiency (up to90%). Subcloning and sequencing confirmed that targeted locus had different mutation types. Furthermore, obvious phenotypes were observed in GO generation after mutation of gsdf. Compared with XY control, histological examination showed that3-month-old Gsdf deficiency XY gonad exhibited partial sex reversal, with oocytes in a small region adjacent to the blood vessel. IHC analysis indicated that Cypl9ala (aromatase, the key enzyme responsible for the production of the17β-estradiol), which was only expressed in interstital cells of the ovary, was found to be expressed in Gsdf deficiency ovotestis. Meanwhile, the expression of Cyp11b2(11β-hydroxylase, the key enzyme responsible for the production of the androgen11-KT) significantly decreased compared with the control XY fish. In addition, blood samples from the Gsdf deficiency and control fish were collected and serum E2and11-KT were measured by EIA. Consistent with the results from IHC, Gsdf deficiency in the XY fish resulted in significantly increased levels of E2and decreased levels of11-KT, compared with the control XY fish. More importantly,6-month-old Gsdf deficiency XY gonad exhibited complete sex reversal displayed a phenotype similar to the normal ovary. Furthermore, the transgenic vector of pIRES-hrGFP-1a-gsdf was constructed and microinjected into XX fertilized eggs at one cell stage. Eight positive transgenic fishes were obtained from30injected fishes by genomic PCR at90dah. The transgenic positive rate was27%. Gonadal histological observations of transgenic positive fishes revealed that the XX gonad exhibited completely sex reversal. Dmrt1and Cyp11b2were found to be expressed in the transgenic positive gonads. In contrast, no Cypl9ala was detected by IHC. These results suggested that Gsdf plays a critical role in the development and maintenance of the testis in tilapia.In summary, tilapia gsdf was isolated from its genome sequences. Sequence homo logy and phylogenetic analyses showed that gsdf was relatively conserved during the fish evolution, gsdf was probably originated from primitive gnathostomata, exists in fishes, such as chondrichthian (elephant shark), and amiacalva (spotted gar), crossopterygii (latimaria), actinopterygians (zebrafish). Gsdf was found to be expressed from5dah, the critical period of tilapia male sex determination and differentiation. The expression level of Gsdf in XY gonads was significantly higher than that in XX gonads. Gsdf deficiency in XY fish resulted in male to female sex reversal, while overexpression of Gsdf in XX fish resulted in female to male sex reversal. Taken together, these results demonstrated that Gsdf is important for male sex determination and the testis maintenance.