Functional diversity and convergence of small non-coding RNAs in male germ cell differentiation and fertilization (mRNA analysis)

The small non-coding RNAs (sncRNAs) are considered as postranscriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs or rRNAs processing may also play important regulatory roles in spermatogenesis. By next generation sequencing (NGS), we characterized the different sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs) at 13.5 dpc, pubertal spermatogonia cells, and mature spermatozoa. In order to assess the potential transmission of the sncRNAs through the mature spermatozoa during fertilization, the sncRNA population detected in male germ cells was also compared with sncRNAs detected in unfertilized mouse oocytes and zygotes. Combining the data obtained by NGS and microarrays from whole PGC and spermatogonia transcriptome, we defined the potential regulatory roles of specific miRNAs and their validated targets. Similar to miRNAs, both the small RNAs derived from snoRNAs and the piRNAs, could be involved in the postranscriptional regulation of mRNA transcripts during the male germ development. Finally, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. These new classes of piRNAs are not generated by the ping-pong pathway and could be the source of primary piRNAs. mRNA analysis of Primordial Germ Cells (PGCs), Spermatogonia cells (SPG), adult testis (AdT) and Gonad-less (GL) embryos. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data.小型非编码RNA（small non-coding RNAs, sncRNAs）被认为是雄性生殖细胞发育过程中的关键转录后调控因子。除微小RNA（microRNAs, miRNAs）与PIWI相互作用RNA（PIWI-interacting RNAs, piRNAs）外，由核仁小RNA（small nucleolar RNAs, snoRNAs）、转运RNA（transfer RNAs, tRNAs）或核糖体RNA（ribosomal RNAs, rRNAs）加工产生的其他小型非编码RNA，也可能在精子发生过程中发挥重要调控作用。本研究通过下一代测序（next generation sequencing, NGS），对雄性生殖细胞分化三个关键里程碑阶段的各类小型非编码RNA群体进行了表征：13.5天胎龄（days post coitum, dpc）的原始生殖细胞（primordial germ cells, PGCs）、青春期精原细胞，以及成熟精子。为评估成熟精子在受精过程中传递小型非编码RNA的潜在可能性，我们将雄性生殖细胞中检测到的小型非编码RNA群体，与未受精小鼠卵母细胞及合子中的小型非编码RNA群体进行了比对分析。结合下一代测序与微阵列技术获取的全原始生殖细胞及精原细胞转录组数据，我们明确了特定微小RNA及其已验证靶标的潜在调控功能。与微小RNA类似，源自核仁小RNA的小型RNA与PIWI相互作用RNA，均可参与雄性生殖细胞发育过程中mRNA转录本的转录后调控。本研究结果强烈提示，源自转运RNA与核糖体RNA的小型RNA可与PIWI蛋白（尤其是MILI）发生相互作用。这类新型piRNA并非通过乒乓通路生成，或可作为初级piRNA的来源。本研究同时对原始生殖细胞（PGCs）、精原细胞（SPG）、成年睾丸（AdT）及无性腺（GL）胚胎的mRNA进行了分析。研究通过多组芯片开展间接比较，将从不同通道获取的原始数据进行整合分析，并与对照数据进行比对。