Potential Contributions of miR-200a/-200b and Their Target Gene-Leptin to the Sexual Size Dimorphism in Yellow Catfish

Abstract

Sexual size dimorphism is the consequence of differential expression of sex-biased genes related to feeding and growth. Leptin is known to regulate energy balance by regulating food intake. In order to investigate the molecular mechanism of sexual size dimorphism in yellow catfish (Pelteobagrus fulvidraco), the expression of leptin (lep) and its functional receptor (lepr) were detected during larval development. Both lep and lepr have lower expression in males than in females during 1-4 weeks post hatching. 17a-Methyltestosterone (MT) treatment resulted in decreased expression of lep and lepr in both male and female larval fish. Interestingly, the mRNA levels of lep and lepr in juvenile male were significantly decreased compared with juvenile female during short-term fasting periods. Lep was predicted to be a potential target of miR-200a and miR-200b that had an opposite expression pattern to lep in male and female larvas. The results of luciferase reporter assay suggested that lep is a target of miR-200a/-200b. Subsequently, male hormone and fasting treatment have opposite effects on the expression of miR-200a/-200b and lep between males and females. In summary, our results suggest that sexual size dimorphism in fish species is probably caused by the sexually dimorphic expression of leptin, which could be negatively regulated by miR-200a/-200b.

Keywords: fasting; leptin; miR-200a/b; sex hormone; sexual dimorphism.

Figure 1

Expression of lep and lepr in juvenile fish among 1–4 weeks post hatching. (A,B) Indicated the relative expression levels of lep and lepr gene during juvenile fish, respectively (^**P < 0.01 and ^***P < 0.001).

Figure 2

Expression of miR-200a and miR-200b in juvenile fish among 1–4 weeks post hatching. (A,B) indicated the relative expression levels of miR-200a and miR-200b during juvenile fish, respectively (^**P < 0.01 and ^***P < 0.001).

Figure 3

Lep is a target of miR-200a. (A) Conserved miR-200a seeding sequence and its binding site in the leptin 3′UTR are marked in red. (B) The sequence information of the putative lep 3′UTR binding site in wild type (WT) and mutant (MUT) were showed. (C) miR-200a suppressed the activity of pmirGLO-lep 3′UTR plasmid, but not mutant vector. The Firefly activity was normalized to Renilla expression, which was used as a control for transfection efficiency. Luciferase assays were performed in triplicate and were representative of 3 independent experiments. This experiment was carried on in Hela cell line. NC, negative control. The asterisk indicates the significant differences of gene expression between different groups (^***P < 0.001).

Figure 4

Effects of fasting on the expression of lep, lepr mRNA and miR-200a/b during juvenile growth in yellow catfish. (A–D) showed the effects of short-term fasting on the expression of lep (A,B) and lepr (C,D) mRNA during growth in female and male juvenile yellow catfish respectively. Fast, fasting group; Control, control group. (E–H) showed the effects of short-term fasting on the expression of lep (E), lepr (F), and miR-200a (G) and miR-200b (H) mRNA in male and female juvenile yellow catfish, respectively. The asterisk indicates the significant differences of gene expression between different groups (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).

Figure 5

Effects of MT on the expression of lep and lepr mRNA during juvenile growth in male and female yellow catfish. (A–D) showed the expression of lep and lepr mRNA respectively. MT, MT treatment group; Control, control group. The asterisk indicates the significant differences of gene expression between the females and males (^**P < 0.01 and ^***P < 0.001).

Figure 6

Different effects of MT on the expression of lep and miR200a/b mRNA during juvenile growth between male and female yellow catfish. (A–C) showed the effects of MT on the expression of lep (A) and miR-200a (B) and miR-200b (C) mRNA in male and female juvenile yellow catfish, respectively. The asterisk indicates the significant differences of gene expression between the females and males (^***P < 0.001).

Figure 7

Model for the regulatory networks of feeding in yellow catfish involved miR-200/leptin. In yellow catfish juvenile, miR-200 regulate feeding by directly repressing leptin gene. Further, miR-200 and ghrelin genes are up-regulated by MT and fast treatment, thereby resulting in the down-regulation of leptin and leptin receptor, thus forming a regulatory network.