Genome-wide differential expression of genes and small RNAs in testis of two different porcine breeds and at two different ages

Abstract

Some documented evidences proved small RNAs (sRNA) and targeted genes are involved in mammalian testicular development and spermatogenesis. However, the detailed molecular regulation mechanisms of them remain largely unknown so far. In this study, we obtained a total of 10,716 mRNAs, 67 miRNAs and 16,953 piRNAs which were differentially expressed between LC and LW pig breeds or between the two sexual maturity stages. Of which, we identified 16 miRNAs and 28 targeted genes possibly related to spermatogenesis; 14 miRNA and 18 targeted genes probably associated with cell adhesion related testis development. We also annotated 579 piRNAs which could potentially regulate cell death, nucleosome organization and other basic biology process, which implied that those piRNAs might be involved in sexual maturation difference. The integrated network analysis results suggested that some differentially expressed genes were involved in spermatogenesis through the ECM-receptor interaction, focal adhesion, Wnt and PI3K-Akt signaling pathways, some particular miRNAs have the negative regulation roles and some special piRNAs have the positive and negative regulation roles in testicular development. Our data provide novel insights into the molecular expression and regulation similarities and diversities of spermatogenesis and testicular development in different pig breeds at different stages of sexual maturity.

Figure 1. Complex of within-breed and inter-breed comparison for differentially expressed genes.

A total of 10,716 unique DEGs (up-regulated and down-regulated genes), 2,645 DEGs are in inter-breed and 10,290 DEGs are in within-breed.

Figure 2. GO term of within-breed comparison of DEGs.

The Top 20 GO (biological process) term analyses of DEGs of LW_a vs LW_b and LC_a vs LC_b.

Figure 3. The expression of miRNAs in LW and LC pigs.

(A) 54 DE miRNAs in LW pigs: 37 up-regulated expressed miRNAs and 17 down-regulated expressed. (B) 43 DE miRNAs in LC pigs: 20 up-regulated expressed miRNAs and 23 down-regulated expressed. Among these DE miRNAs, 14 were co-upexpressed and 16, meanwhile 16 were co-downexpressed.

Figure 4. Concise functional interaction network of selected TDETGs.

The effect of the interaction is represented by arrows, straight line and imaginary line. “− >” for activating/catalyzing, “−” for FIs extracted from complexes or inputs, and “−” for predicted FIs.

Figure 5. Integrated network of miRNA–mRNA–piRNA.

Comparison between before and after sexual maturity in within-breed. DDU: down-regulated piRNA and down-regulated mRNA and up-regulated miRNA; UDU: up-regulated piRNA and down-regulated mRNA and up-regulated miRNA, “− >” for activating, “−” for inhibition.

Figure 6. Validation of DE miRNAs and TDETGs by Q-PCR.

Histograms of the relative expression levels of a/b (a: 200 days mature testes; b: 20 days immature testes) from LW and LC pigs. The x-axis represents the DE miRNAs and TDETGs, and the y-axis is the fold-change between the two groups (log(−ΔΔCt values, 2) for Q-PCR, log(a/b, 2) for sequencing). In total, the QPCR results have consisted with RNA-Seq results, three TDETGs (COL6A2, ITGB1 and CD34) and four DE miRNAs (miR-10b, miR-148a-3p, miR-181d-5p, miR-181a) were down-regulated; four TDETGs (SPATA24, KHDBPS3, TSGA10, GGNBP) and one DE miRNA (miR-133a-3p) were up-regulated by two methods. Although the relative expressions of two genes (CCNI and NEURL) and three miRNAs (miR-145-5p, miR-301 and miR-194b-5p) have different patterns using Q-PCR and RNA-Seq, the direction of expression is same by two methods.

Figure 7. Integrated network analysis of RNA and miRNA.

Analyses depend on within-breed comparison and results are in both LW and LC pigs. Different colors represent different biological processes, and similar biological processes are classified into one category, and seven major categories are exist.