| MicroRNAs (miRNAs) are endogenous small RNAs that can regulate target mRNAs by binding to their sequences in the 3’untranslated region (UTR). These have been declared to be involved in a variety of functions, including developmental transitions, neuronal patterning, apoptosis, adipogenesis metabolism and hematopoiesis in different organisms. This study is focussed on the expression of miRNAs and the biogenetic pathways are involved in sexual differentiation and the regulation of the development of primordial germ cells and gonadal somatic cells.Sexual transformation in rice field eel (Monopterus albus) occurs naturally from female to male during its life cycle. To study the physiological mechanism of its sex reversion,160 mature miRNA sequences were obtained from the brain, liver and gonad of the rice field eel of different sexual stages (female, intersex and male) based on the high-throughput sequencing technology. These miRNAs were considered as highly expressed miRNA in the rice field eel, including Let-7 family, miR-10b, miR-143, miR-26a, miR-21, miR-181a and miR-9. A significantly differential expression among the 3 stages of rice field eel gonad development (P< 0.001) was observed for 48 unique miRNAs. Only 9 unique miRNAs showed a more than 8-fold change in their expression among the 3 stages, including miR-430 family. The target mRNA of miR-430 was predicted and the interaction network of target genes was analysed by Ingenuity Pathway Analysis (SPA). Most targets of miR-430 were associated with the reproductive system development.To investigate the role of miR-430 family during the fish sex reverse, we chose the Japanese medaka (Oryzias latipes) as the experimental model in fish sex determination and differentiation. The different expression of miR-430 family members gradually enlarged between adult male and female medaka of brain-liver-gonad. At the age of 5 dpf (days post fertilization), male and female fertilize eggs were exposed to the sex hormones (17β-Estradiol or 17β-Trenbolone) medium for 7 days. The expression of ola-miR-430b was increased after exposure to the either sex hormones. Meanwhile, we predicted the regulation relationship of miR-430 family and target gene foxl2 in medaka and rice field eel, and validated by dual luciferase assay. The result showed that mal-miR-430b was binding to the foxl 2 3’UTR of the rice field eel. However, medaka foxl2 gene avoids some miRNAs such as miR-430 family regulation. It may be due to rapid 3’UTR that are specifically depleted of miRNA binding sites. Therefore, we speculate that by the miR-430 family did regulate to Foxl2 that would affect the natural sex reversal in rice field eel.Forkhead family phylogeny suggests that duplicated forms of Foxl2 which are named Foxl2a (Foxl2) and Foxl2b (Foxl3) have been existed in different teleost species. We hypothesized that it is the existence of copy foxl3 in the rice field eel. Through RACE method cloning, the ORF (open reading frame) of foxl3 full length cDNA from rice field eel were 795 bp that codes for a protein 264 amino acids. The rice field eel Foxl3 amino acid sequences exhibited high identity with the European sea bass (Dicentrarchus labrax,82.9%). Phylogenetic tree was built for 11 teleosts based on Forkhead family amino acid sequences using ML (Maximum Likelihood) method. Three major clusters (Foxll, Foxl2 and Foxl3) were recognized. The Foxl3 amino acid sequence of rice field eel, zebrafish (Danio rerio), rainbow trout (Oncorhynchus mykiss), Japanese medaka (Oryzias latipes) and European sea bass were clustered into one branch, which had a closer genetic relationship with European sea bass (bootstrap 89). The foxl3 tissues distribution and differential expression in gonad were analyzed by semi-RT-PCR and real-time qPCR. Elevated levels offoxl3 expression were also observed in the spleen, kidney and brain. And foxl3 mRNA was less expressed in blood and heart tissues. The foxl3 expression level was increased during the natural sex-reverse of the rice field eel. The relative expression in the testis is greater than 40 times than that in the ovary (P<0.05). The foxl3 expression trend during the sex-reverse of the rice field eel was matched with transcriptome data. Dual luciferase assay validated that the miR-9 not miR-430a binding to foxl3 3’UTR of the rice field eel. We speculated that the foxl3 was regulated by miR-9 after transcription in rice field eel. More research is needed on the Foxl3 regulation, particularly as it may be a new way to the study of rice field eel natural sex reversal. |
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