Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis

Spermatogenesis is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N6-methyladenosine (m6A), an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m6A mRNA methylomes of mouse spermatogenic cells from five developmental stages: undifferentiated spermatogonia, type A1 spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactiva- tion of the m6A RNA methyltransferase Mettl3 or Mettl14 with Vasa-Cre causes loss of m6A and depletion of SSCs. m6A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Com- bined deletion of Mettl3 and Mettl14 in advanced germ cells with Stra8-GFPCre disrupts spermiogenesis, whereas mice with single deletion of either Mettl3 or Mettl14 in advanced germ cells show normal spermatogenesis. The sper- matids from double-mutant mice exhibit impaired translation of haploid-specific genes that are essential for spermio- genesis. This study highlights crucial roles of mRNA m6A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis. Undifferentiated spermatogonia, type A1 spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids were isolated from wild-type mice (B6 background ) to perform m6A-seq. Undifferentiated spermatogonia from 5-day-old wild-type, Mettl3-vKO and Mettl14-vKO mice were isolated to perform ribosome profiling and RNA-seq. Pachytene spermatocytes and round spermatids from wild-type and Mettl3/14-sKO were isolated to perform ribosome profiling and RNA-seq.