AGO4 Regulates Entry into Meiosis and Influences Silencing of Sex Chromosomes in the Male Mouse Germline

The four mammalian Argonaute family members are thought to share redundant functions in the microRNA pathway, yet only AGO2 possesses the catalytic "slicer" function required for RNA interference. Whether AGO1, AGO3, or AGO4 possess specialized functions remains unclear. Here, we Series_summary = show that AGO4 localizes to spermatocyte nuclei during meiotic prophase I, specifically at sites of asynapsis and in the transcriptionally silenced XY sub-domain, the sex body. We generated Ago4 knockout mice and show that Ago4-/- spermatogonia initiate meiosis early, resulting from premature induction of retinoic acid-response genes. During prophase I, the sex body assembles incorrectly in Ago4-/- mice, leading to disrupted meiotic sex chromosome inactivation (MSCI). This is associated with a dramatic loss of microRNAs, >20% of which arise from the X chromosome. Loss of AGO4 results in increased AGO3 in spermatocytes, indicating some degree of redundancy. Thus, AGO4 regulates meiotic entry and MSCI in mammalian germ cells, implicating small RNA pathways in these processes. mRNA transcripts were isolated and prepared using pachytene spermatocytes, pre-meiotic testes and other tissues from Ago4+/+ and Ago4-/- littermates and sequenced using Illumina HiSeq2000. small RNA transcripts were isolated and prepared using pachytene spermatocytes from adult Ago4+/+ and Ago4-/- littermates and sequenced using Illumina GAII.哺乳动物Argonaute家族的四个成员被认为在微小RNA（microRNA）通路中存在功能冗余，但仅AGO2具备RNA干扰所需的催化"slicer"功能。目前尚不清楚AGO1、AGO3与AGO4是否具有特异性功能。本研究中，我们发现AGO4在减数分裂I前期定位于精母细胞核内，特异性定位于联会异常位点以及转录沉默的XY亚结构域——性体（sex body）。我们构建了Ago4基因敲除小鼠，研究显示Ago4全敲除（Ago4-/-）的精原细胞会提前启动减数分裂，该表型由视黄酸应答基因的过早诱导所导致。在减数分裂I前期，Ago4-/-小鼠的性体组装异常，进而引发减数分裂性染色体失活（meiotic sex chromosome inactivation，MSCI）紊乱。该表型与微小RNA的显著缺失相关，其中超过20%的微小RNA来源于X染色体。AGO4的缺失会导致精母细胞内AGO3的表达水平升高，提示二者存在一定程度的功能冗余。综上，AGO4可调控哺乳动物生殖细胞的减数分裂启动与MSCI，表明小RNA通路（small RNA pathways）参与了上述过程。我们从Ago4野生型（Ago4+/+）与Ago4敲除型（Ago4-/-）同窝小鼠的粗线期精母细胞、减数分裂前睾丸组织及其他组织中分离并制备mRNA转录本，使用Illumina HiSeq2000平台进行测序。同时，我们从成年Ago4+/+与Ago4-/-同窝小鼠的粗线期精母细胞中分离并制备小RNA转录本，使用Illumina GAII平台进行测序。