Mettl3-/Mettl14-mediated mRNA N⁶-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 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 inactivation 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. 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 m⁶A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.