Table_2_Analysis of Long Non-Coding RNA and mRNA Expression Profiling in Immature and Mature Bovine (Bos taurus) Testes.xlsx

Testis development and spermatogenesis are strictly regulated by numbers of genes and non-coding genes. However, long non-coding RNAs (lncRNAs) as key regulators in multitudinous biological processes have not been systematically identified in bovine testes during sexual maturation. In this study, we comprehensively analyzed lncRNA and mRNA expression profiling of six bovine testes at 3 days after birth and 13 months by RNA sequencing. 23,735 lncRNAs and 22,118 mRNAs were identified, in which 540 lncRNAs (P-value < 0.05) and 3,525 mRNAs (P-adjust < 0.05) were significantly differentially expressed (DE) between two stages. Correspondingly, the results of RT-qPCR analysis showed well correlation with the transcriptome data. Moreover, GO and KEGG enrichment analyses showed that DE genes and target genes of DE lncRNAs were enriched in spermatogenesis. Furthermore, we constructed lncRNA–gene interaction networks; consequently, 15 DE lncRNAs and 12 cis-target genes were involved. The target genes (SPATA16, TCF21, ZPBP, PACRG, ATP8B3, COMP, ACE, and OSBP2) were found associated with bovine sexual maturation. In addition, the expression of lncRNAs and cis-target genes was detected in bovine Leydig cells, Sertoli cells, and spermatogonia. Our study identified and analyzed lncRNAs and mRNAs in testis tissues, suggesting that lncRNAs may regulate testis development and spermatogenesis. Our findings provided new insights for further investigation of biological function in bovine lncRNA.

睾丸发育与精子发生受到大量编码基因与非编码基因的严格调控。然而，作为众多生物过程关键调控因子的长链非编码RNA（long non-coding RNAs，lncRNAs），在公牛性成熟过程中的睾丸组织内尚未得到系统性鉴定。本研究通过RNA测序技术，对出生后3日龄与13月龄的6份公牛睾丸组织的lncRNA与mRNA表达谱进行了全面分析。最终共鉴定出23735条lncRNA与22118条mRNA，其中540条lncRNA（P值<0.05）与3525条mRNA（校正P值<0.05）在两个发育阶段间呈显著差异表达（differentially expressed，DE）。相应地，实时荧光定量PCR（reverse transcription quantitative polymerase chain reaction，RT-qPCR）分析结果与转录组数据呈现良好的相关性。此外，基因本体（Gene Ontology，GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析结果显示，差异表达基因以及差异表达lncRNA的靶基因均显著富集于精子发生通路。进一步构建了lncRNA-基因互作网络，最终涉及15条差异表达lncRNA与12个顺式靶基因。经鉴定，上述靶基因（SPATA16、TCF21、ZPBP、PACRG、ATP8B3、COMP、ACE及OSBP2）均与公牛性成熟过程相关。此外，在公牛睾丸间质细胞（Leydig cells）、支持细胞（Sertoli cells）与精原细胞（spermatogonia）中均检测到了lncRNA及其顺式靶基因的表达。本研究鉴定并分析了睾丸组织中的lncRNA与mRNA，提示lncRNA可能参与调控睾丸发育与精子发生过程。本研究结果为进一步探究公牛lncRNA的生物学功能提供了全新的研究视角。