Mettl3-/Mettl14-mediated mRNA N(6)-methyladenosine modulates murine spermatogenesis.

Spermatogenesis is a differentiation process during which diploid spermatogonial stem cells (SSCs) produce haploid spermatozoa. This highly specialized process is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N(6)-methyladenosine (m(6)A), an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m(6)A mRNA methylomes of mouse spermatogenic cells from five developmental stages: undifferentiated spermatogonia, type A(1) spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactivation of the m(6)A RNA methyltransferase Mettl3 or Mettl14 with Vasa-Cre causes loss of m(6)A and depletion of SSCs. m(6)A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Combined 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 spermatids from double-mutant mice exhibit impaired translation of haploid-specific genes that are essential for spermiogenesis. This study highlights crucial roles of mRNA m(6)A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.