Transcriptome Analysis Of XY,YY Yellow Catfish Testes And Functional Study Of Rbmx In Fish Gonad Development

Yellow catfish(Pelteobagrus fulvidraco)is an important economic freshwater fish,widely distributes in China.All-male yellow catfish has been applied in production,whereas its biological mechanism of sex differentiation and gonad development has remained elusive.The XY male and YY super-male yellow catfish provide an ideal model for studying the male sex determination and differentiation of testis in fish species.In our study,XY and YY testes were sequenced by Illumina high-throughput platform.A total of 78148 unigenes were identified,in which 1146 unigenes have significantly higher expression in YY testis and 1235 unigenes have significantly higher expression in XY testis.Among them 605 differently expressed unigenes were annotated into 1604 GO items with 286 and 319 unigenes having relative higher expression in YY and XY testis respectively.KEGG enrichment analysis found that 312 differently expressed unigenes were annotated into 252 signal pathways.Several differently expressed unigenes were related to PI3K-AKT and G protein-coupled receptor(GPCR)signal pathways.Real-time fluorescent quantitative PCR identified 12 differently expressed unigenes and the results were consistent with high-throughput sequencing.Furthermore,miR-141/429 were speculated to play key function in testis development,bioinformatic analysis predicted that some factors in PI3K-AKT and GPCR signaling pathways were potential targets of miR-141/429,several of which were confirmed by dual-luciferase reporter assays system.In our study,a comparative transcriptome analysis was performed between XY and YY testis,which revealed that the interaction of miR-141/429 and their targets plays important function in testis development and spermatogenesis.Interestingly,bioinformatic prediction found that rbmx is the target of miR-141/429.qRT-PCR results indicated that rbmx has significantly higher expression in YY testis than in XY testis,which is contrary to the expression of miR-141/429.Previous studies have shown that RBMX and its retrogenes are highly expressed in testis and indispensable for testis development and spermatogenesis in mammals.Due to a relatively long reproductive cycle and lacking of genome information in yellow catfish,rbmx functional research was performed in zebrafish.The retrogenes of RBMX are only existed in mammals,and our phylogenetic analysis showed that the functional differentiation of RBMX retrogenes has been established during early evolution,RBMX and RBMY appeared as sex chromosome formation,and positive selection was identified in the branch of mammals.However,only one copy of rbmx gene was identified in bonyfish and its expression and function remains unknown.Spatiotemporal expression analysis showed that rbmx gene is highly expressed in the brain and liver in adult zebrafish and mainly expresses in brain during zebrafish embryogenesis.We constructed rbmx knockout system by CRISPR/Cas9.Finally,the homozygous mutant zebrafish with 2nt deletion was obtained.A higher male-proportion has been observed in offspring of rbmx mutants,which may due to a female to male sexual reversal during rbmx mutants development.Furthermore,a significantly lower sperm motility has also been observed in rbmx mutants.The results showed that rbmx plays important role in sex differentiation and gamete formation in fish.Our study provided an important living material and new avenues for research in Illuminating gonad development and gametogenesis of bonyfish.