Role of Mir-145 and Mir-181 in Nile Tilapia (Oreochromis niloticus) Sex Differentiation

Nile tilapia is an important fish species due to its relevance to worldwide aquaculture. Male specimen have increased growth when compared with females, an important feature in fish production systems. Unfortunately the mechanisms lead to sex differentiation in fish are still poorly understood. Several genes were proposed as triggers of sex differentiation and miRNAs were also described as important players in this process. Considering the biological importance of sex determination we have analyzed Nile tilapia miRNomes focusing on miRNAs that are expressed in male and female during the development. Small RNA libraries were generated from 3 and 5 dpf embryos, and adult male and female brains and gonads and sequenced on an Illumina platform. We identified 194 miRNAs, of these 120 showed differential expression between male and female and 74 were novel miRNAs. Three miRNAs were tested by qPCR and the 3’UTRs of candidate target genes were tested by luciferase reporter assay. qPCR results showed that miR-145 expression increased during development of female embryos and its putative target gene, Sox9, decreased. On the other hand, expression of miR-181 decreased and its putative target gene, Cyp191a, increased. The reporter assays validate Cyp19a1 as a direct target of miR-181, and Sox9 as a direct target of miR-145. The target genes have relevant miRNA binding sites in their 3'UTR and mutation of the binding site renders the reporter construct insensitive to the miRNA. Our data suggest miR-145 and miR-181 are important in male and female differentiation, respectively, by regulating SOX9 and Cyp19a1 genes.