Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish

Abstract

Although evolutionary fates and expression patterns of duplicated genes have been extensively investigated, how duplicated genes co-regulate a biological process in polyploids remains largely unknown. Here, we identified two gsdf (gonadal somatic cell-derived factor) homeologous genes (gsdf-A and gsdf-B) in hexaploid gibel carp (Carassius gibelio), wherein each homeolog contained three highly conserved alleles. Interestingly, gsdf-A and gsdf-B transcription were mainly activated by dmrt1-A (dsx- and mab-3-related transcription factor 1) and dmrt1-B, respectively. Loss of either gsdf-A or gsdf-B alone resulted in partial male-to-female sex reversal and loss of both caused complete sex reversal, which could be rescued by a nonsteroidal aromatase inhibitor. Compensatory expression of gsdf-A and gsdf-B was observed in gsdf-B and gsdf-A mutants, respectively. Subsequently, we determined that in tissue culture cells, Gsdf-A and Gsdf-B both interacted with Ncoa5 (nuclear receptor coactivator 5) and blocked Ncoa5 interaction with Rora (retinoic acid-related orphan receptor-alpha) to repress Rora/Ncoa5-induced activation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a). These findings illustrate that Gsdf-A and Gsdf-B can regulate male differentiation by inhibiting cyp19a1a transcription in hexaploid gibel carp and also reveal that Gsdf-A and Gsdf-B can interact with Ncoa5 to suppress cyp19a1a transcription in vitro. This study provides a typical case of cooperative mechanism of duplicated genes in polyploids and also sheds light on the conserved evolution of sex differentiation. Polyploidy generates extra chromosome sets and duplicated genes. However, how the duplicated genes co-regulate a biological process in polyploids remains largely unknown. Here, we reveal that two gsdf (gonadal somatic cell-derived factor) homeologs (gsdf-A and gsdf-B) cooperatively induce male differentiation by inhibiting cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) transcription in hexaploid gibel carp (Carassius gibelio). Loss of either gsdf-A or gsdf-B alone results in partial male-to-female sex reversal and loss of both causes complete sex reversal, which can be rescued by a nonsteroidal aromatase inhibitor. Compensatory expression of gsdf-A and gsdf-B is observed in gsdf-B and gsdf-A mutants, respectively. Moreover, we determine that in vitro, Gsdf-A and Gsdf-B both interact with Ncoa5 (nuclear receptor coactivator 5) and block Ncoa5 interaction with Rora (retinoic acid-related orphan receptor-alpha) to repress Rora/Ncoa5-induced activation of cyp19a1a. These findings reveal the potential molecular mechanisms underlying gsdf homeologs-mediated male differentiation in polyploid fish.