| Sexual dimorphism is a common phenomenon in animals. Although there are clear morphological, behavioral and physiological differences between females and males, both sexes share mostly identical genomes. Thus, differential gene expression in both sexes may contribute to sexual dimorphism. miRNAs involve as one type of post-transcriptional regulators in sexual dimorphism. Gonads are the primary organs in sexual reproduction that produce both gametes and steroid hormones. Due to divergent differentiation pathways, the testis and ovary demonstrate obvious morphological dimorphism. Thus, they are often chosen as target tissues for investigating the mRNA and miRNA expressions. Therefore, it is important to study the genome-wide defferentially expressed genes between testis and ovary, and to investigate the molecular mechanism of the difference.Nile tilapia, Oreochromis niloticus, is one of the most commonly farmed species in freshwater aquaculture. Because of the male growing faster than the female by nearly 30%, The male fish farming has better economic value. Tilapia is a good experimental animal for the study of sex determination and differentiation, due to short sexual maturation time and spawning cycle. In addition, tilapia whole genome sequencing has been completed and fully open, which has the good genome resources. Therefore, the research results from tilapia are useful for both basic research (mechanism for sex determination)and aquaculture (sex manipulation).In this study, we use both molecular and bioinformatics methods to profile miRNA and mRNA expressions in testes and ovaries of young Nile tilapia aiming at discovering the possible mechanisms responsible for sexual dimorphism in gonads. At the same time, we also analyze the influence of estradiol and methyltestosterone on miRNA expression, and explore the mechanism that miRNAs mediated the regulation negulation network in fish sex determination. The main results were as follows:(1) The study of sexual dimorphism in mRNA expression profile from gonads of Nile tilapiaYoung Nile tilapias at 60 days post hatch (dph) from six fish for each sex were used to construct two RNA libraries of ovaries and testes, and mRNA-Seq libraries were sequenced on the Illumina sequencing platform. The result showed that:16909 and 13495 genes expressed in testis and ovary, respectively. Seminal plasma glycoprotein, heat shock protein HSP 90-beta-like,40S ribosomal protein and 60S ribosomal protein were high expression genes in testis. ubiquitin-conjugating enzyme E2 C-like, histone Hloo-like, Fatty acid binding protein, FABP and gametocyte-specific factor 1-like were high expression genes in ovary. These highe expression genes in testis and ovary represented different developmental pathway between gonads.5559 differentially expressed genes were obtained, in which,2299 genes were up-regulated and 3260 genes down-regulated in ovary. Pathway analysis revealed that a lot of differentially expressed genes were enriched in metabolic processes, at the same times, many differentially expressed genes were involved in reproductive biology pathways.(2) The study of sexual dimorphism in miRNA expression profile from gonads of Nile tilapiaTwo RNA libraries of ovaries and testes were sequenced on the Illumina sequencing platform. After trimming of adaptor sequences and removal of the low-quality sequences, a total of 13030780 and 26675871 clean reads in the ovary and testis libraries were obtained from miRNA-Seq data, respectively. A total of 152 differentially expressed miRNAs were detected, in which, expression differences between sexes of 76 conserved miRNAs were significant. By comparing,67 up-regulated and nine down-regulated miRNAs were detected in ovary. There were a lot of SNPs in miRNA expression profile. Some SNPs were co-expressed in two gonads; some of them were specifically expressed in testis or ovary. The differentially expressed miRNAs and specifically expressed SNPs may contribute to the formation of sexual dimorphism.(3) Coordinated miRNA/mRNA expression profiles of gonads from Nile tilapiaThe target genes of differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Furthermore, KEGG pathway analyses were conducted in these coincident genes. By miRNA-mRNA correlating and computational target predicting, two types of negatively regulatory miRNA-mRNA correlations (up-or down-regulated miRNA and down-or up-regulated mRNA) were obtained. Seven functional miRNA-target gene pairs, miR-17-5p/Dmrtl, miR-20a/Dmrtl, miR-138/Cyp17a2, miR-338/Cyp17a2, miR-200a/Cyp17a2, miR-456/Amh and miR-138/ Amh, were predicted at the sequence level and further detected by real-time PCR based on the significantly negative relationships. These differentially expressed miRNAs suppressed gene expressions of gender and then prometed the formation of sexual dimorphism in Nile tilapia.(4) The influence of estradiol and methyltestosterone on miRNA expression from gondas of Nile tilapiaJuvenile Nile tilapias were dealt with estradiol and methyltestosterone, and 8 differently expressed miRNAs (miR-122?miR-214?miR-202?let-7b?let-7h?miR-17-5p?miR-200 and miR-143) in tesitis and ovary were studied. The result showed that these miRNAs were higher expressed in ovary than testis. Estradiol caused the expression of 8 miRNAs in testis and ovary greatly reduced. After Methyl testosterone processing, miRNA expression in ovary increased and decreased after a period time, and in testis decreased at all the time. At the same time, the correlation was analyzed between the expression of 8 miRNAs and three genes (Cyp19a1a, Foxl2 and Dmrt1). The result showed that expressions of 8 miRNAwere correlated with that of Cyp19a1a and Foxl2 significantly, and negatively correlated with that of Dmrtl significantly, and the expression of these miRNA were correlated with each other significantly. The steroid hormone influced the miRNA expression related to sex, and then affected sexual dimorphism of Nile tilapia. |
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