The miRNA expression profiles of round spermatids during GRTH/DDX25 mediated spermiogenesis: mRNA-miRNA Network analysis [mRNAseq]. The miRNA expression profiles of round spermatids during GRTH/DDX25 mediated spermiogenesis: mRNA-miRNA Network analysis [mRNAseq]

GRTH/DDX 25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. Regulation of spermatogenesis occurs at the levels of transcription, post-transcriptional and translational levels which needs to be explored in detail. miRNAs regulate the expression of important genes involved in spermatid elongation process. Differential miRNA expression in round spermatids (RS) of GRTH KO and GRTH-KI mice in comparison with mRNAs remains unexplored. Our miRNAseq analysis of small RNAs obtained from RS reveal differential expression of important miRNAs that regulate spermatid differentiation, apoptosis, chromatin compaction and ubiquitination. mRNAseq analysis of RS isolated from GRTH-KI and GRTH-KO mice models were also carried-out to study the putative miRNA targets and mRNA-miRNA interaction. mRNAseq analysis revealed specific set of mRNAs that are critical for sperm chromatin compaction, ubiquitination and spermatid elongation etc. miRNA-mRNA interaction prediction highlighted differentially expressed/enriched miRNA transcripts whose predicted mRNA targets were differentially expressed. Based on the interaction information of the miRNAs-mRNAs differential analysis, the miRNA-mRNA network was constructed. These results demonstrate the importance of miRNA in the translational arrest and stability of germ cell specific mRNAs which are critical for later stages of spermiogenesis and fertility. Overall design: Differential expression analysis of mRNA and miRNA transcriptomic profile in round spermatids. This study compares the miRNA/mRNA profiles of GRTH knock-out and GRTH knock-in round spermatids to wild-type to assess the role of GRTH in spermiogenesis.

GRTH/DDX25是DEAD-box家族RNA解旋酶（DEAD-box family of RNA helicases）的一员，在精子发生过程中发挥至关重要的作用。精子发生的调控可发生于转录、转录后及翻译三个层面，该过程仍有待深入探究。微小RNA（microRNA，简称miRNA）可调控参与精子细胞伸长过程的关键基因表达。相较于信使RNA（messenger RNA，简称mRNA），GRTH敲除（GRTH knock-out，简称KO）与GRTH敲入（GRTH knock-in，简称KI）小鼠的圆形精子细胞（round spermatids，简称RS）中差异表达的miRNA仍未得到系统研究。本研究对从RS中分离得到的小RNA开展miRNA测序（miRNA sequencing）分析，发现了一批可调控精子细胞分化、细胞凋亡、染色质浓缩及泛素化过程的差异表达关键miRNA。同时，本研究对从GRTH-KI与GRTH-KO小鼠模型中分离得到的RS进行mRNA测序（mRNA sequencing）分析，以探究推定的miRNA靶基因及miRNA-mRNA互作关系。mRNA测序结果显示，存在一组与精子染色质浓缩、泛素化及精子细胞伸长等过程密切相关的特征性mRNA。miRNA-mRNA互作预测分析表明，存在一批差异表达/富集的miRNA转录本，其预测的mRNA靶基因同样呈现差异表达特征。基于miRNA-mRNA差异分析得到的互作信息，本研究构建了miRNA-mRNA调控网络。上述结果证实了miRNA在生殖细胞特异性mRNA的翻译阻滞与稳定性维持中发挥的重要作用，而该过程对于精子形成的后期阶段及生育能力至关重要。
实验整体设计：对圆形精子细胞中的mRNA与miRNA转录组谱进行差异表达分析。本研究将GRTH敲除与GRTH敲入小鼠的圆形精子细胞的miRNA/mRNA谱与野生型小鼠进行对比，以评估GRTH在精子发生过程中的作用。