Abstract
The present study aims to reveal the mechanism by which miR-430s regulate steroidogenesis in larval rice field eel Monopterus albus. To this end, M. albus embryos were respectively microinjected with miRNA-overexpressing mimics (agomir430a, agomir430b, and agomir430c) or miRNA-knockdown inhibitors (antagomir430a, antagomir430b, and antagomir430c). Transcriptome profiling of the larvae indicated that a total of more than 149 differentially expressed genes (DEGs) were identified among the eight treatments. Specifically, DEGs related to steroidogenesis, the GnRH signaling pathway, the erbB signaling pathway, the Wnt signaling pathway, and other pathways were characterized in the transcriptome. We found that steroidogenesis-related genes (hydroxysteroid 17-beta dehydrogenase 3 (17β-hsdb3), hydroxysteroid 17-beta dehydrogenase 7 (17β-hsdb7), hydroxysteroid 17-beta dehydrogenase 12 (17β-hsdb12), and cytochrome P450 family 19 subfamily a (cyp19a1b)) were significantly downregulated in miR-430 knockdown groups. The differential expressions of miR-430 in three gonads indicated different roles of three miR-430 (a, b, and c) isoforms in regulating steroidogenesis and sex differentiation. Mutation of the miR-430 sites reversed the downregulation of cytochrome P450 family 17 (cyp17), cyp19a1b, and forkhead box L2 (foxl2) reporter activities by miR-430, indicating that miR-430 directly interacted with cyp17, cyp19a1b, and foxl2 genes to inhibit their expressions. Combining these findings, we concluded that miR-430 regulated the steroidogenesis and the biosynthesis of steroid hormones by targeting cyp19a1b in larval M. albus. Our results provide a novel insight into steroidogenesis at the early stage of fish at the molecular level.
Highlights
Steroidogenesis plays pivotal roles in regulating physiological divergence, gonadal differentiation, and sex determination [1]
The potential functions and metabolic pathways of differentially expressed genes (DEGs) were further analyzed by Toenrichment explore the and potential effect of miR-430 on steroidogenesis, we investigated using Gene ontology (GO)
Mutation of the miR‐430 binding sites in 3′ UTR of cyp17, found in liver, followed by heart, ovary, muscle, intestine, spleen, and the lowest was cyp19a1b, and foxl2 completely abolished the inhibition of luciferase reporter activities by found in kidney, withthe noregulatory differences among the miR‐430s tissues ofonhead kidney, brain, testis, and miR‐430, validating effects of the the expression of cyp17, ovotestis (Figure 4G). These results revealed that miR-430a and miR-430b exhibited the highest expression in ovotestis among three gonads, and miR-430c displayed a higher expression in ovary than in ovotestis and testis
Summary
Steroidogenesis plays pivotal roles in regulating physiological divergence, gonadal differentiation, and sex determination [1]. L2, foxl; steroidogenic factor 1, sf-1; Wilms tumor 1, wt1) have been found to be involved in the sex determination process [9,10,11]. Cytochrome P450 family 19 subfamily a (cyp19a) has been identified to transform androgens into estrogens, which can be regulated by foxl together with other partners sf-1 [12,13]. Cytochrome P450 family 17 (cyp17) as the steroid synthase transcriptional activator has been reported to be negatively regulated by the interaction of foxl and sf-1 in granulosa cells [14]. Some key genes have been identified, the critical network in sex steroid hormone synthesis as well as reproductive processes is still unclear and needs to be further investigated
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