High-Resolution Profiling of Novel Transcribed Regions During Rat Spermatogenesis

Mammalian spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-coding transcriptome, little effort has yet been devoted to the systematic identification of novel unannotated transcribed regions expressed during mammalian spermatogenesis. We report high-resolution expression profiling of male germ cells in the rat using Illumina next-generation sequencing technology and highly enriched testicular cell populations. Among 20,424 high-confidence transcripts reconstructed, we defined a stringent set of 1,419 long multi-exonic unannotated transcripts expressed in the testis, named TUTs. These were classified into seven groups with different expression patterns. The vast majority of TUTs share many of the characteristics of vertebrate long non-coding RNAs (lncRNAs). We also markedly reinforced the finding that TUTs and known lncRNAs accumulate during the meiotic and postmeiotic stages of spermatogenesis in mammals, and that X-linked meiotic TUTs do not escape the silencing effects of meiotic sex chromosome inactivation. Importantly, we discovered that TUTs and known lncRNAs with a peak expression during meiosis define a distinct class of non-coding transcripts that exhibit exons twice as long as those of other transcripts. Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource of novel loci expressed during spermatogenesis that significantly contributes to the rat genome annotation. A graphical display of the data is conveniently accessible through the ReproGenomics Viewer (RGV) at http://rgv.genouest.org.哺乳动物精子发生是一系列复杂且高度协调的生理过程，在此过程中雄性生殖细胞通过增殖与分化最终产生成熟精子。尽管近年来的全基因组研究已推动了雄性生殖细胞编码蛋白转录组的深入解析，但针对哺乳动物精子发生过程中表达的新型未注释转录区域的系统性识别，相关研究仍较为匮乏。本研究利用Illumina下一代测序技术与高度富集的睾丸细胞群体，对大鼠雄性生殖细胞开展了高分辨率表达谱分析。在重构得到的20424条高可信度转录本中，我们经严格筛选，划定了1419条在睾丸中表达的长链多外显子未注释转录本，将其命名为TUTs。这些转录本可根据其表达模式划分为7个类别。绝大多数TUTs均具备脊椎动物长链非编码RNA（long non-coding RNAs，lncRNAs）的诸多特征。本研究进一步支持了此前的研究结论：哺乳动物精子发生的减数分裂及减数分裂后阶段中，TUTs与已知lncRNAs的表达水平显著上调；且X染色体连锁的减数分裂期TUTs无法逃逸减数分裂性染色体失活带来的沉默效应。尤为重要的是，本研究发现，在减数分裂期达到表达峰值的TUTs与已知lncRNAs构成了一类独特的非编码转录本，其外显子长度是其他转录本外显子的两倍。本研究为雄性生殖细胞的转录组分析提供了全新视角，同时构建了一套高质量的精子发生过程中表达的新型基因座资源，可为大鼠基因组注释提供重要支撑。研究数据的可视化展示可通过ReproGenomics Viewer（RGV）便捷获取，访问网址为http://rgv.genouest.org。