Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation

Abstract

Background: MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia.

Results: We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish.

Conclusions: The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Keywords: Early sex differentiation; Tilapia; mRNA; miRNA.

Fig. 1

Genomic distribution of identified pre-miRNAs in tilapia

Fig. 2

Comparisons of the number of pre-miRNAs of Oryzias latipes, Ictalurus punctatus, Danio rerio and Salmo salar in miRbase with the number of pre-miRNAs identified in O. niloticus

Fig. 3

Length distribution of miRNA sequences in ovary and testes of tilapia by Illumina small RNA deep sequencing. Sequence length distribution of clean reads based on the abundance and distinct sequences; the most abundant size class was 22 nt, followed by 23 nt and 21 nt

Fig. 4

Circos circular visualization of the miRNA reads coverage on tilapia genome

Fig. 5

Comparison of expression levels of miRNAs in XX and XY gonads. The X axis and Y axis show expression level of miRNAs in the two samples, respectively. Red points represent miRNAs with ratio > 2; green points represent miRNAs with 1/2 < ratio ≤ 2; blue points represent miRNAs with ratio ≤ 1/2. Ratio = normalized expression of XX gonad/normalized expression of XY gonad

Fig. 6

miRNA-gene network in the steroid hormone biosynthesis pathway [48] and regulatory network in early sex differentiation of Nile tilapia. The genes in boxes are differentially expressed in the mRNA-Seq and were validated by previous transcriptomic analyses [72]. Blue box nodes represent upregulated genes in XY gonads, red box nodes represent upregulated genes in XX gonads, and black box nodes represent genes with no significant difference between XX and XY. miRNAs in the ellipses potentially regulated the corresponding gene expression. Blue ellipses represent downregulated miRNAs in XX gonads; red ellipses represent downregulated miRNAs in XY gonads