The Bioinformatical Analysis On Sox Family And Possible Role Of Sox9a/b In The Development Of Nile Tilapia Gonad

Sox (SRY-related HMG-box) genes encode a family of transcription factors that belong to a super-family of genes characterized by a homologous sequence called the HMG (high mobility group) box. Members of this family have been shown to be conserved during evolution and play key roles in a wide variety of developmental processes, including chondrogenesis, hemopoiesis, the development of the nervous system, lens formation and sex determination and differentiation. To date,10and40Sox genes have been indentified in invertebrates and vertebrates (20in mammals,24in fugu and15in medaka), respectively. Based on differing levels of structural, organizational similarities and other features, Sox genes can be subdivided into11groups, named from A to K. Studies on the functions of Sox genes, especially in sex determination and differentiation, were focused on mammals, while a few were reported in fish. In order to study the possible roles of Sox family members in sex determination and differentiation of teleost, we isolated and analyzed all Sox genes in Nile tilapia (Oreochromis niloticus) from genome sequences and5,30,90and180dah (days after hatching) XX and XY gonad transcriptome databases. On the other hand, studies had shown that Sox9and Sox9a/b played important roles in sex determination and differentiation in mammals and medaka, respectively. Therefore, the possible role of Sox9a/b was also investigated in the gonadal development of tilapia.In the present study, we isolated27members in Nile tilapia by homology research using transcriptome and genome sequences. These Sox genes can be devided into seven subfamilies:B(B1:Soxla、Soxlb、Sox2、Sox3、Sox19;B2:Sox14a、Sox14b、Sox21), C(Sox4a、Sox4b、Soxlla、Sox11b)、D (Sox5、Sox6a、Sox6b、Sox13)、E(Sox8a、 Sox8b、Sox9a、Sox9b、Sox10a、Sox10b),F (Sox7、Sox17、Sox18), H (Sox30) and K (Sox32). The HMG domain motif RPMNAF is highly conserved, except for Sox30and Sox32(RPLNAF). Furthermore, Sequence alignment showed that the motif in tilapia Sox30is different from that of the other species, and the motif in Sox32is identical in all species. Through the analysis transcriptomes of Nile tilapia, we found that20Sox genes were expressed in gonads and10of them were obviously sexual dimorphic: Sox3、Sox9a、Sox11a and Sox11b with higher expression in XX gonads, while Sox4a、 Sox4b、Soc7、Sox9b、Sox17and Sox30with higher expression in XY gonads.Gene structure and sequence analyses showed that Sox9is highly conserved during evolution. Tissue distribution analysis by RT-PCR demonstrated that both Sox9a and Sox9b were expressed in the brain, pituitary, gill, head kidney, heart, liver, and muscle in adult tilapia. Sox9a was not detected in the gonads, while Sox9b was only detected in the testis of adult tilapia. The expression profiles of Sox9a/b during gonadal development were examined by real-time PCR. The results were consistent with the results of transcriptome analysis. Promoter analysis by luciferase assays also showed that Sox9a suppressed Sf1-activated Cypl9ala, encodes the key enzyme for estrogen synthesis, activity in a dose-dependent manner when cotransfected with Sfl. Disruption of Sox9a/b was also performed in Nile tilapia using TALENs. TALENs could effeiciently target Sox9a. Histological observations of the3-month-old Sox9a deficiency testes revealed that they developed abnormally with most germ cells degenerated, with a few or even without any spermatogonia, spermatocytes, spermatids and sperms. Proliferations of somatic cells were also detected in some of Sox9a deficiency testes. Immunohistochemistry analysis showed that there were less Amh、 Cyp11b2and Dmrtl positive cells and weak positive signals in Sox9a deficiency testes compared with control testes, indicating fewer Leydid and Sertoli cells in the former.In summary, we identified27Sox members which belong to8subfamilies in Nile tilapia genome. Twenty of them were expressed in gonads and ten of them showed sexul dimorphic expression. Sox9b may be involved in the regulation of estrogen synthesis, while Sox9a may play an important role in testicular development and maintance of germ cell numbers in teleosts.