Identification of a circRNA-miRNA-mRNA network to explore the effects of circRNAs on Holstein bull testis after sexual maturity

Spermatogenesis is an extremely sophisticated and complex process and is regulated not only by a large number of genes, but also by a large number of epigenetic factors. Although existing studies have demonstrated that circRNAs plays an important regulatory role in spermatogenesis, there is still insufficient information to properly understand the regulatory role and mechanism of circRNA action. We addressed this issue by examining the testes of two Holstein bull develop-mental stages; three 8-week-olds (young bull, YB) and three 80-week-olds (adult bull, AB), randomly selected from the same breeding stock. A total of 3032 circRNAs, 683 miRNAs were identified as significantly differentially expressed noncoding RNAs, and 14,081 mRNAs. Based on these results, a circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory network was constructed containing 3298 targeted regulatory axes. Modular analysis revealed a total of four modules in the ceRNA regulatory network. Functional analysis of these results showed that the ceRNA regulatory network in AB testis exhibited more positive regulatory effects on the spermatogenesis cycle checkpoints, chromosome and cytoplasm segregation, sperm tail forma-tion, and sperm motility. In addition, screening combining the results of our previous studies on lncRNA regulation of spermatogenesis revealed 4 genes (FOXO4, PPP1CB, CDC26, and CDKN1B) that co-exist in the 2 ceRNA regulatory networks, lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA. A ceRNA regulatory network was constructed based on these genes. This study demon-strated the possible regulatory role of circRNAs in adult testicular spermatogenesis based on constructed transcriptome profiles and furtzher broadened our understanding of the regulatory role of circRNAs in spermatogenesis.