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

www.nature.com/scientificreports OPEN received: 06 January 2016 accepted: 10 May 2016 Published: 27 May 2016 Genome-wide differential expression of genes and small RNAs in testis of two different porcine breeds and at two different ages Yao Li1,*, Jialian Li1,2,*, Chengchi Fang1, Liang Shi3, Jiajian Tan3, Yuanzhu Xiong1, Bin Fan1,2 & Changchun Li1 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. Small RNAs (sRNA, 19–35 nucleotides in size) are derived from diverse genome contexts, which involve intergenic regions, transposable elements and their remnants, or originate from viruses1–3 and non-coding regulatory molecules with diverse functions in various pathways4,5. sRNAs play critical roles in direct mRNA degradation6, translational repression7, heterochromatin formation8 and DNA elimination9. Currently, at least three types of sRNAs have been described: microRNAs (miRNAs) which are generated from hairpin-shaped precursors10, small interfering RNAs (siRNAs) which originate from double-stranded RNAs (dsRNAs)11 and PIWI-interacting RNAs (piRNAs) which are expressed specifically and abundantly in spermatogenic cells5. Recent studies have indicated that sRNAs including miRNA12–14 and piRNA15,16 play important roles in the regulation of gene expression related to spermatogenesis or/and testicular development. In detail, over-expression of miR-34c, in the later stages of spermatogenesis, can down regulate the TGIF2 gene, a repressor of the TGF-β signaling pathway which is crucial for spermatogenesis17. MiR184, miR21 and miR449 are associated with the functional regulation of gonadal somatic cells; miRNAs play diverse roles in the development and physiology of gonadal cells in mammalian reproduction18. Mael129, a conserved piRNA pathway protein Maelstrom, is associated with piRNA precursors and exhibits the reduced translation of numerous spermatogenic mRNAs, including those encoding acrosome and flagellum proteins19. Testicular development and spermatogenesis are the major processes in male reproduction20. Germ cells are the direct participants in spermatogenesis, which is the key in reproduction. Spermatogenesis is a complex process through which diploid germ cells proliferate and differentiate into haploid spermatozoa21. This complex 1 Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China. 2Guangxi Yangxiang Pig Gene Technology limited Company, Guigang, 537120, People’s Republic of China. 3Guangxi Yangxiang Incorporated Company, Guigang, 537100, People’s Republic of China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to C.L. (email: ) Scientific Reports | 6:26852 | DOI: 10.1038/srep26852 1 www.nature.com/scientificreports/ process of cell differentiation is controlled by many factors and fundamentally orchestrated through the expression of thousands of protein-encoding genes, which are developmentally regulated during spermatogenesis and play pivotal roles during specific phases of germ cell development. Previous studies have identified 1,652 spermatogenesis-related genes in the developing testis; 351 of these genes are expressed only in the male germ cells, with germ cell-specific transcripts being much less common earlier in testicular development22. Some genes for normal sperm are also indispensable. For example, the absence of ADAM2 can lead to abnormal functions of sperm23. Tnp2 translation is necessary for sperm maturation and male fertility24. Inversely, Max represses stem cell development25. Numerous genes are more significantly expressed in meiotic and/or early haploid spermatogenic cells than in somatic cells but inefficiently translated26. Existing evidence suggests the fluctuating expression of genes at different testicular development stages and the differential expression of genes in various testicular cells. During the testicular development and spermatogenesis, well-ordered and sequential changes in gene expression in each cell type are required to create a fully functional testis which is capable of producing mature spermatozoa27, necessary for sexual maturation. Lu Chuan (LC) boars derived from Guangxi Zhuang Autonomous Region in China are characterized by a small body size and early sexual maturity; conversely, Large White (LW) boars derived from Canada feature a large body size, rapid growth and late sexual maturity. LC and LW boars considerably differ in sexual maturation time; LW boars reach maturity at ~180 days old28, whereas LC boars reach maturity at 90 days old29. Spermatogenesis is an important factor determining sexual maturation time and process. Previous studies proved that miRNA30 or piRNA31 are present in pig testes at sexual immaturity and maturity. However, the similarities and diversities of genes and sRNA, as well as their regulatory relationship, in the spermatogenesis and testicular development of different pig breeds at different maturity stages remain unclear. In the present study, Illumina GAIIx sequencing technology was employed to profile miRNAs, piRNAs and mRNAs in the testes of LC and LW boars at sexual immaturity and maturity to investigate differentially expressed miRNAs, piRNAs and genes related to spermatogenesis and testicular development. The results of this study indicate that specific sRNAs and genes play important regulatory roles in testicular maturation, and contribute to sexual maturation differences between pig breeds. Results To obtain a comprehen (...truncated)