| Two closely related tilapiine species, the blue tilapia(Oreochromis aureus) and Nile tilapia (O. niloticus), with high similarity between their genome sequence, adopted ZW and XY sex determination system, respectively. The fact that inter-specific hybridization between the two fishes produces fertile offspring, suggests that there is a coordination mechanism between the two different regulating networks of sexual determination systems. These have made them an excellent model system for analyzing the genetic and molecular mechanisms underlying the evolution of different mode of sex determination. Transcriptomic analyses play critical role in understanding the relationship between similar genome sequence and different sex-determining systems. Therefore, a comparison of the gonadal transcriptomes of closely related tilapiine species should provide valuable data on the evolution of diverse sex-determination mechanisms. It is well known that estrogen and androgen play important roles in sex determination and maintenance. The comparative analysis of steroidogenic enzyme and steroid receptors genes, which belong to nuclear receptor (NR) superfamily, is helpful to understand the sexual regulation network. The NR superfamily, which is divided into 7 subfamilies, constitutes one of the largest classes of transcription factors and plays important roles in many significant physiological and biochemical processes, especially in sex determination and differentiation. However, the evolution, expression and function of NRs in vertebrates have not been fully understood yet. In the present study, we performed comparative transcriptome analyses of gene expression profile in the gonads of two closely related tilapiines, explored the relationship between NR superfamily expansion and animal genome duplication, and analyzed the spatial and temporal expression profile of NRs. The results were as follows:Transcriptomes of ZW and ZZ gonads from the blue tilapia at 180 days after hatching (dah) were sequenced using Illumina HiseqTM technology, which produced 16 Gb sequences and mapped to 20,710 genes. Of these,17,720 genes were expressed in both ZW and ZZ gonads,125 genes were ZW-specific and 2,866 genes were ZZ-specific. Genes with "FDR≤0.01", "|log2 (ZW_RPKM/ZZ_RPKM)|≥1" and "total RPKM>4"were identified as ZW or ZZ up-regulated genes, while genes with "|log2 (XX_RPKM/XY_RPKM)|<1" or "FDR≥0.01" and "total RPKM>4"were identified as co-expressed genes (COG). In total, there are 114,634,3516,4395 and 6470 genes were ZW-SEGs, ZZ-SEGs, ZW-DIGs, ZZ-DIGs and COGs, respectively. These results were consistent with previous reports in the Nile tilapia, zebrafish and Xenopus, which suggested that the physiological processes of the testis may require more genes than the ovary.Comparative analysis of gonadal transcriptome between the blue tilapia and Nile tilapia showed that 2548 genes were up-regulated in female (ZW and XX),3359 genes were up-regulated in male (ZZ and XY) and 4194 genes were co-expressed in gonads of both two tilapiines. The results suggested that these genes were associated with gonadal phenotype rather than genotype or sex-determining system. The expression profiles of almost all steroidogenic enzyme genes and steroid receptor genes in the male and female gonads of the blue tilapia were consistent with those in the Nile tilapia, suggesting that the expression of these genes were phenotype-dependent rather than genotype-dependent. The expression profiles of Cyp19a1a, Cyp11b2, StAR1 and StAR2, in the gonads of the two tilapias at 180 dah detected by IHC (immunohistochemistry) were consistent with those by transcriptomic analyses. In contrast, there were still some genes showed distinct expression profiles in the two tilapiines, which may be genotype dependent and associated with different sex-determining systems.In this study, through comprehensive database search, we identified all NRs (including 4 novel members) from the tilapia (75), common carp (137), zebrafish (73), fugu (73), tetraodon (72), stickleback (70), medaka (69), coelacanth (55), spotted gar (51) and elephant shark (50). For 21 NRs, two duplicates were found in teleosts, while only one in tetrapods. These duplicates, except those of DAX1, SHP and GCNF found in the elephant shark, were derived from 3R (the third round of genome duplication). The linkage duplication of 5 syntenic blocks (comprising 14 duplicated NR couples) in teleosts further supported their 3R origin. Based on transcriptome data from 8 tissues of adult tilapia,53 NRs were found to be expressed in more than one tissue. Of them,10 genes were expressed ubiquitously in all eight tissues. COUP-TFa, ERRS, ERa, SF-1, EAR2-B, RXRy and Rev-erbAβ-B were expressed at the highest level in the brain, heart, liver, testis, kidney, muscle, head kidney and ovary, respectively, which indicated their essential roles in the corresponding tissue. Based on the XX and XY gonadal transcriptome data from four developmental stages,65 NRs were found to be expressed in the gonads, with 21,31,11 and 29 sexually dimorphic NRs at 5,30,90 and 180 dah, respectively. The expression of four selected genes was examined by ISH and qPCR to validate the spatial and temporal expression profiles of NRs. Similar expression profiles were observed in ISH, qPCR and transcriptome data. Comparative analyses of the expression profiles of duplicated NRs revealed divergence in gene expression as well as gene function.Taken together, this work provided a new perspective on the evolution and function of NR superfamily and valuable data on the study of sex determination, differentiation and maintenance in teleosts, and laid the foundation for studying the molecular evolution of different mode of sex determination in closely related tilapiines. |
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