Mi Ou,Kunci Chen,Dandan Gao,Yanduo Wu,Qing Luo,Haiyang Liu,Jian Zhao
Dmrt1 is an important transcriptional regulator that plays critical role in male gonadogenesis, testicular differentiation and development. In this study, Dmrt1 was cloned from blotched snakehead ( Channa maculata ) , which is designated as CmDmrt1 . Cm Dmrt1 encoded a putative protein with 293 amino acids and presented an extremely conserved DM domain. It was nearly expressed in the gonads, and the expression was more than 15 times higher in the testis than in the ovary. 1851 bp promoter sequence of CmDmrt1 was characterized and the methylation levels of the CpG sites were analyzed to detect sex-related differences. A significant negative correlation between CmDmrt1 expression and CpG methylation level of its promoter was found in the testis and ovary. During gonadal development, CmDmrt1 transcription displayed strong male-biased expression patterns, increased with the maturation of testis and reached the peak at 195 days after hatching (dah), which indicates a significant role of Dmrt1 in spermatogenesis. Steroid treatment could influence CmDmrt1 expression, and long-term 17β-estradiol (E2) treatment could induce the male-to-female secondary sex reversal (SSR), which resulted in the differentiated testis transformed to ovary or ovotestis. Meanwhile, CmDmrt1 expression was down-regulated to fairly low level in the ovary of the SSR XY fish, which was similar to that in normal XX females ovary. Our research illustrates that Dmrt1 is linked to testis differentiation and spermatogenesis in blotched snakehead, providing information for functional studies on sex differentiation and gonadal development of C. maculata , and scientific basis for the production practice of all-male snakehead breeding.
The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 ( cyp17a1 )-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L ( fancl ) expression in the gonads of doublesex and mab-3 related transcription factor 1 ( dmrt1 )-/- and cyp17a1 -/-; dmrt1 -/- fish. The additional depletion of fancl in cyp17a1 -/-; dmrt1 -/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1 -/-; dmrt1 -/-; fancl -/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro . The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1 -/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.
Gonadal sex differentiation is known as the queen of problems in evolutionary biology, and the mechanisms that determine sexual fate vary widely among teleosts. Traditionally, Dmrt1 and androgen signaling have been essential for testis differentiation, and estrogen signaling has been essential for ovary differentiation. The all-testis phenotype observed in cyp17a1 -/- zebrafish, has led to the conclusion that androgen signaling is dispensable for testis differentiation in zebrafish. By analyzing a series of mutant zebrafish lines, we show here that Dmrt1 sufficiently promotes all-testis differentiation in cyp17a1 -deficient zebrafish albeit the compromised androgen and estrogen synthesis. In addition, we observed that zebrafish Dmrt1 and androgen signaling probably stabilize Tp53 by inhibiting the transcription of a ubiquitin ligase, Fancl. Our current study provides new insights into the interactive signals that regulate sexual fate determination in teleosts.
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