Deregulated sex chromosome gene expression with male germ cell-specific loss of Dicer1 (miRNA array data)

MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs that mediate post-transcriptional gene silencing by inhibiting mRNA translation and promoting mRNA decay. DICER1, an RNAse III endonuclease encoded by Dicer1, is required for processing short 21-22 nucleotide miRNAs from longer double-stranded RNA precursors. Here, we investigate the loss of Dicer1 in mouse postnatal male germ cells to determine how disruptions in the miRNA biogenesis pathway may contribute to infertility. Reduced levels of Dicer1 transcripts and DICER1 were confirmed in germ cell knock-out (GCKO) testes by postnatal day 18 (P18). Compared to wild-type (WT) at 8 weeks, GCKO males had no change in body weight, yet showed significant reductions in testis mass and sperm number. Histology and fertility tests confirmed spermatogenic failure in GCKO males. Array analyses at P18 showed 96% of miRNA genes were down-regulated and 37% of protein-coding genes were differentially expressed in GCKO testes. Interestingly, we observed preferential overexpression of genes on the sex chromosomes in GCKO testes, with more than 80% of the genes overlapping those proposed to undergo meiotic sex chromosome inactivation (MSCI) in the germ cells. Compared to WT, GCKO mice showed higher percentages of cells at early meiotic stages (leptotene and zygotene) but lower percentages at later stages (pachytene, diplotene and metaphase I), providing evidence that deletion of Dicer1 leads to disruptions in meiotic progression. Furthermore, we observed fewer elongating spermatids with proper translational activation of transition protein 2 (Tnp2), protamine 1 and 2 (Prm1 and Prm2) in GCKO testes after step 12-14. Therefore, deleting Dicer1 in early postnatal germ cells causes misregulation of transcripts encoded by genes on the sex chromosomes, impairs meiotic progression and post-meiotic translational control and results in spermatogenic failure and infertility. Total RNA, including miRNAs, were purified from a total of six individual mouse samples. The tissue collected was obtained from wild-type (control; n=3) and Dicer1 germ cell knockout (mutant; n=3) testes on P18. One miRNA GCKO sample, M36, was determined to be of poor quality and was excluded from the study; thus, a total of five miRNA samples were analyzed.微小RNA（MicroRNAs，miRNAs）是一类内源性非编码RNA，通过抑制mRNA翻译并促进mRNA降解介导转录后基因沉默。由Dicer1基因编码的核糖核酸酶III内切核酸酶DICER1，是从较长双链RNA前体加工生成21~22核苷酸长度短miRNA所必需的因子。 本研究以小鼠出生后雄性生殖细胞中Dicer1的缺失为模型，探究miRNA生物合成通路异常如何导致不育。研究人员在出生后第18天（P18）的生殖细胞敲除（germ cell knock-out，GCKO）小鼠睾丸中，证实了Dicer1转录本及DICER1蛋白水平均显著降低。 与8周龄野生型（WT）小鼠相比，GCKO雄性小鼠体重无明显变化，但睾丸重量与精子数量均出现显著下降。组织学检测与生育力实验证实，GCKO雄性小鼠存在生精障碍。 P18阶段的芯片分析结果显示，GCKO小鼠睾丸中96%的miRNA基因发生下调，37%的蛋白编码基因呈现差异表达。值得注意的是，本研究观察到GCKO小鼠睾丸中性染色体上的基因呈现优先过表达趋势，其中超过80%的基因与已报道的生殖细胞减数分裂性染色体失活（MSCI）相关基因重合。 与野生型小鼠相比，GCKO小鼠早期减数分裂阶段（细线期与偶线期）的细胞占比更高，而晚期减数分裂阶段（粗线期、双线期与第一次减数分裂中期）的细胞占比更低，这一结果证明Dicer1缺失会破坏减数分裂进程。此外，在GCKO小鼠睾丸的第12~14阶段后，可观察到发生正常翻译激活的过渡蛋白2（Tnp2）、鱼精蛋白1和2（Prm1与Prm2）的阳性伸长型精子细胞数量减少。 综上，在出生后早期生殖细胞中敲除Dicer1会导致性染色体编码基因的转录本表达失调，损害减数分裂进程与减数分裂后翻译调控，最终引发生精障碍与不育。 本研究共收集6份小鼠样本以提取包括miRNA在内的总RNA，样本取自P18阶段的野生型（对照组，n=3）与Dicer1生殖细胞敲除（突变组，n=3）小鼠睾丸。其中1份miRNA样本M36经检测质量不佳，被排除出本研究，最终共纳入5份miRNA样本进行分析。