New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis

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.