Expression dynamics, relationships, and transcriptional regulations of diverse transcripts in mouse spermatogenic cells

Among all tissues of the metazoa, the transcritpome of testis displays the highest diversity and specificity. However, its composition and dynamics during spermatogenesis have not been fully understood. Here, we have identified 20,639 message RNAs (mRNAs), 7,168 long non-coding RNAs (lncRNAs) and 15,101 circular RNAs (circRNAs) in mouse spermatogenic cells, and found many of them were specifically expressed in testes. lncRNAs are significantly more testis-specific than mRNAs. At all stages, mRNAs are generally more abundant than lncRNAs, and linear transcripts are more abundant than circRNAs. We showed that the productions of circRNAs and piRNAs were highly regulated instead of random processes. Based on the results of a small-scale functional screening experiment using cultured mouse spermatogonial stem cells, many evolutionarily conserved lncRNAs are likely to play roles in spermatogenesis. Typical classes of transcription factor binding sites are enriched in the promoters of testis-specific m/lncRNA genes. Target genes of CREM and RFX2, 2 key TFs for spermatogenesis, were further validated by using ChIP-chip assays and RNA-seq on RFX2-knockout spermatogenic cells. Our results contribute to the current understanding of the transcriptomic complexity of spermatogenic cells and provide a valuable resource from which many candidate genes may be selected for further functional studies.

在后生动物的所有组织中，睾丸的转录组（transcriptome）展现出最高的多样性与特异性。然而，精子发生（spermatogenesis）过程中转录组的组成及其动态变化尚未被完全阐明。本研究在小鼠生精细胞中鉴定出20639条信使RNA（messenger RNA，mRNA）、7168条长链非编码RNA（long non-coding RNA，lncRNA）与15101条环状RNA（circular RNA，circRNA），并发现其中诸多分子仅在睾丸中特异性表达。长链非编码RNA的睾丸特异性显著高于信使RNA。在所有发育阶段，信使RNA的表达丰度普遍高于长链非编码RNA，线性转录本的表达丰度也高于环状RNA。本研究证实，环状RNA与PIWI蛋白互作RNA（piwi-interacting RNA，piRNA）的生成过程受到严格调控，而非随机事件。基于利用培养小鼠精原干细胞（spermatogonial stem cell）开展的小规模功能筛选实验结果，诸多进化保守的长链非编码RNA可能在精子发生中发挥功能。睾丸特异性mRNA/lncRNA基因的启动子区域富集有典型的转录因子结合位点（transcription factor binding site）。本研究进一步通过染色质免疫共沉淀芯片（chromatin immunoprecipitation-chip，ChIP-chip）实验以及针对RFX2敲除生精细胞的RNA测序（RNA sequencing，RNA-seq），验证了精子发生关键转录因子CREM与RFX2的靶基因。本研究结果增进了学界对生精细胞转录组复杂性的现有认知，并提供了一项宝贵的研究资源，可从中筛选诸多候选基因用于后续功能研究。