Characterization Of Gonadal Transcriptomes From Nile Tilapia(Oreochromis Niloticus) And Bioinformatic Analysis Of Fox Gene Family

Sex determination and maintenance in fish involved complex processes with many interacting events. Females and males differentiated in opposite directions with combined actions of chromosomal and environmental factors. The Nile tilapia (Oreochromis niloticus) is a world wide cultured fish with the male growing faster than female. China is by far the main tilapia producing and exporting country. Thus, investigation of the molecular mechanisms of sex determination and maintenance will provide a theoretical basis for the production of all-male tilapia and is of great significance in aquaculture.Up to now, the majority of studies on sex determination and differentiation have been focusing on single gene. The emergence of transcriptome, is a breakthrough technology for researches in this area. Illumina HiseqTM technology was used to sequence four pairs of XX and XY gonads from Nile tilapia at four developmental stages:1)5days after hatching (5dah), the critical time for sex determination and differentiation;2)30dah, initiation of germ cell meiosis and oogenesis in XX gonads;3)90dah, initiation of germ cell meiosis or spermatogenesis in XY gonads; and4)180dah, sperm maturation and vitellogenesis in XY and XX gonads. The sequences were mapped to21,334genes in tilapia genome. Of these,21,006genes were found to be expressed in both XX and XY gonads,259and69genes were found to be specifically expressed in XY and XX gonads, respectively. Intrestingly, more genes were found to be expressed in XY gonads than in XX gonads at all four developmental stages. In this study,"FDR≤10-2" and "|log2(XX_RPKM/XY_RPKM)≥1" were used to identify XX and XY up-regulated genes. XX and XY up-regulated genes expressed at5dah and in at least two other developmental stages in XX and XY gonads were considered XX and XY-enhanced genes, respectively. Totally,187and1,358genes were identified as XX-and XY-enhanced genes. Genes expressed in both XX and XY gonads with "FDR>10-2" and "|log2(XX_RPKM/XY_RPKM)|<1" at all four developmental stages were considered co-expressed genes (COG).2,978genes were identified as COG. The expression levels of almost all steroidogenic enzyme genes (including cypl9ala and cypl9alb, genes encoding the key enzyme aromatase for estrogen synthesis) were found to be higher in XX than in XY gonads at5and30dah. Nearly all the steroidogenic enzyme genes (including of cyp11b2, encoding11β-hydroxylase, the key enzyme for androgen synthesis in tilapia) were significantly up-regulated at90dah. At180dah, the expression profiles of almost all steroidogenic enzymes in XY gonads were further increased, while that in XX gonads remain unchanged. Our results indicated the XX fish produced estrogen and the XY fish produce neither estrogen nor androgen at5dah. Both estrogen receptors (esrl, esr2a and esr2b) and androgen receptors(ar1and ar2) were found to be expressed in XX gonads, while only estrogen receptors (esr2a and esr2b) were expressed in XY gonads at5dah. This could explain why exogenous androgen and estrogen treatment induced XX and XY sex reversal. The XX-enhanced expression of cypl9ala and cypl9alb at all stages suggests an important role for estrogen in female sex determination and maintenance of phenotypic sex.Forkhead transcription factors play important roles in regulating many important cell processes including development, cell proliferation, differentiation, stemness maintenance, stress response, language acquirement, and longevity. Hundreds of Fox genes have been identified in eukaryotic organisms ranging from yeasts to humans, and have been classified into19subfamilies (A to S) according to the amino sequence of their conserved forkhead domains. Up to now, only four members have been identifies in fungus, while45genes have been identified in mammals. The remarkablely increased members may indicate there was complicated expansion of genes during the evolutionary process. Thus, we reconstructed the phylogenetic relationships of Fox family members to reveal the expanding and evolutionary process. Here we isolated67Fox members, belonging to18subfamilies, from the Nile tilapia genome and transcriptome sequences. Probably due to the species specific duplication of Foxml,5Foxml members were found in Nile tilapia. Further analysis indicated Foxsl and Foxc2were absent in tilapia and other teleost genomes. The presence of68Fox members in teleosts was the result of three times of whole genome duplication (3R). Transcriptome analysis showed that52Fox members were expressed in tilapia gonadal transcriptomes. Most of them showed sexual dimorphic expression pattern.Taking together, this work provided the largest collection of gonadal transcriptome data in tilapia. The transcriptome data futher confirmed the critical roles of estrogen in sex determination and maintenance. The co-expressive and XX/XY-enhanced genes identified in this study laid the basis for the elucidation of the molecular mechanism involved in these processes. In this study, we also identified all Fox members from Nile tilapia and the other animals, and analyzed their phylogenepic relationship. Most of the52Fox members, which were expressed in tilapia gonadal transcriptomes, were sexual dimorphically expressed in tilapia gonads, suggesting that Fox family members may play important roles in tilapia sex determination, differentiation, and maintenance of phenotypic sex.