Genetic Analyses Of Sex-related Genes During Gonadal Differentiation And Sexual Phenotype Formation In Olive Flounder, Paralichthys Olivaceus

In the present study, we first cloned olive flounder (Paralichthys olivaceus) dmrt1, dmrt4 and sox9 genes. Together with olive flounder P450arom gene, their expression patterns in adult tissues were analyzed using convertional RT-PCR. The result identified these four genes'sexual dimorphic expression patterns. We further studied the expression of these genes during flounder gonadal differentiation and development through real-time RT-PCR and in situ hybridization. We also expressed flounder Dmrt1 fusion protein in vitro using E.coli BL-21(DE3) expression system. Meanwhile, we studied the methylation patterns of these four genes'promoter CpG island in testis and ovary at the epigenetic level.Flounder dmrt1's total cDNA sequence is 3373bp, and its binding sites of sex-related transcriptional factors Sry, Sox9 and Sox5 were identified in the promoter region. The encoded protein contains a conserved DM domain. Flounder dmrt4 gene included two exons and one intron, of which promoter region contained binding site of sex-related transcriptional factor Sox9. A conserved DM domain and a specific DMA domain were found in the encoded protein. Flounder sox9 gene included three exons and two introns, and binding sites of transcriptional factors Ftz, Foxd3 and Oct1 were identified in its promoter region. This gene encoded a protein containing a conserved HMG domain. The 5'UTR of flounder P450arom was cloned, in which a DM domain binding site, two steroidogenic factor 1 (Sf1) binding sites and a forkhead-responsive consensus site (Foxl2) binding site were identified.The tissue specific expression patterns of dmrt1, dmrt4, sox9 and P450arom in adult flounder indicated that these four genes were all sex-related genes with sexual dimorphic expression. In detail, flounder dmrt1's expression was restricted to gonads, and was higher expressed in testis than that in ovary. Both dmrt4 and sox9 were stronger in testis and weaker in ovary, and were also differentially expressed in some other tissues. P450arom was higher expressed in ovary than that in testis, and was expressed in some other tissues with varied degrees too. Results of real-time RT-PCR indicated that flounder dmrt1 was scarcely expressed in primitive gonad and during following periods of gonadal differentiation. But its expression went up rapidly in differentiating testis, and remained very low in differentiating ovary. Flounder dmrt4 was strongly expressed in primitive gonad, and much lower expressed during following periods of gonadal differentiation. Then its expression became stronger in differentiating testis. The sox9 was also highly expressed in flounder primitive gonad, much lower expressed after that and had some fluctuated expression during gonadal differentiation period. And then, it differentially expressed in differentiating testis and ovary. However, P450arom expressed in primitive gonad at first, and its expression quantity went upward and downward during following periods of gonadal differentiation. In the early stage of differentiating ovary, its expression became stronger.These four genes are also differentially expressed during five developmental stages of gonad. Flounder dmrt1 was weakly expressed in the testis and ovary of stage I, and its expression quantity went up rapidly in the testis of stage II. After that, the quantity went a little down in the testis of stage IV. Then its expression quantity reached the peak in the testis of stage V. From the stage II to the stage V, the expression quantity in ovaries were all scare. The dmrt4 gene was highly expressed in the testes of stage I and II with upward trend. Then its expression quantity went down significantly in the stage III and IV, but went up a little until stage V. The expression quantities in all the five stages of ovaries were always low. Flounder sox9 had lower expression quantity in the testis of stage I than in the ovary of stage I. And then, its expression in testis began to go up. From the stage II to V, the expression quantities were higher in testes than that in ovaries. Its expression quantities in ovary reached the peak in the stage II. From the stage III to V, there was almost no expression in the ovaries. P450arom gene had higher expression quantity in the testes from stage I to V than thoes in the ovaries. The difference of quantity was bigger in the stage I and II, in which the expression quantities of ovaries were very high. And after that, the expression in ovary went down. But its expression in testis was always weak.Gonad tissue slice in situ hybridization results showed that transcripts of dmrt1, dmrt4 and sox9 were mainly localized in spermatocytes and Sertoli cells of flounder testis, and the signal in spermatocytes was stronger than that in Sertoli cells, but there was weak signal detected in ovary. On the contrary, flounder P450arom mRNA was located in follicular cells and oocytes of ovary, and the signal in follicular cells was stronger than that in oocytes, and there was only a little signal detected in spermatocytes of testis. Whole mount in situ hybridization analysis showed that during flounder embryogenesis, flounder dmrt4 is expressed in the ectodermally derived olfactory placodes, neuroectodermal forebrain and telencephalon and otic placodes during embryogenesis. The expression level of dmrt4 is always high in the olfactory placodes for all these stages from neurula stage to the hatching stage, but it is dynamic in the telencephalon. While transcripts are distributed throughout the forebrain at early stages, remaining strongly expressed from the neurula stage to tail-bud forming stage, the expression domain becomes more and more restricted to a small area around the first ventricle in the dorsal telencephalon and is relatively weak at the early tail-bud stage.Results of genes'promoter CpG islands methylation patterns analyses showed that there were sexual dimorphic differences in the CpG methylation level of flounder dmrt1,dmrt4和P450arom promoters. No CpG demethylation of flounder dmrt1 promoter in testis and its 57.69% CpG island (CGI) methyaltion in ovary corresponded to this gene's about 70 times higher expression in testis than that in ovary. On the other hand, 97.5% CGI methylation of flounder P450arom promoter in testis and its 73.33% CGI methyaltion in ovary correlated to this gene's about 40 times lower expression in testis than that in ovary. The levels of flounder dmrt4 promoter CGI methylation in gonads were also sexual dimorphic. There were 2% CGI methylation of this gene promoter in testis and 7% methylation in ovary, which was consistent with its sexual dimorphic expression with higher expression in testis than that in ovary. However, there was no difference to be detected in the levels of flounder sox9 promoter CGI methylation in both gonad, and its sox9 promoter CpGs in testis and ovary were all totally demethylated, which was inconsistent with this gene's differential expression pattern with higher expression in testis than that in ovary. Flounder de novo DNA (cytosine-5)-methyltransferases 3 (dnmt3) had a sexual dimorphic expression pattern that was higher expressed in testis than that in ovary. And it was also differentially expressed in kidney, spleen, brain and eye. Dnmt3 was highly expressed in flounder primitive gonad, and had some fluctuated expression during gonadal differentiation period. It was sexually dimorphic expressed during differentiation and development of testis and ovary.Flounder dmrt1 gene (encoded 293aa protein with molecular weight of 32.5 KDa) was recombined and fused on plasmid pPROEXTMHTa, and was transferred into E.coli BL-21(DE3) for Dmrt1 expression in vitro. The result of SDS-PAGE showed that fusion protein highly expressed inside the E.coli BL-21(DE3) cell after induction. Most fusion protein expressed in the form of inclusion bodies, and only a little fusion protein expressed in the form of soluble protein. The fusion Dmrt1 protein had the highest expression quantity after 4 h induction with 0.6mmol/L IPTG. When the induction temperature was 34℃, expression quantity of the soluble protein was the maximum.