The dynamic and reversible regulation roles of m 6 A modification, and the characterization of m 6 A readers have provided new insights into spermatogenesis at post-transcriptional level. YTHDF2 has been reported to recognize and mediate the m 6 A-containing transcripts decay during the mouse oocyte mature, embryonic stem cell differentiation, neural development, and zebrafish maternal-to-zygotic transition. However, the roles of YTHDF2 in mammalian spermatogenesis are uncertain. Here, we generated germ cell-specific Ythdf2 mutants ( Ythdf2 -vKO) at a C57BL/6J background, and demonstrated that YTHDF2 was essential for mouse spermatogenesis and fertility. Ythdf2 -vKO provided oligoasthenoteratozoospermia(OAT) phenotype with increased apoptosis in germ cells. High-throughput RNA-seq of the testis tissue showed the failure of the degradation of a wave of YTHDF2 target mRNA. Interestingly, RNA-seq analysis combined with our previous single-cell transcriptomics data of mouse spermatogenesis pointed out the failure of a wave of transcript transition during the spermatogenesis of Ythdf2 -vKO, which was confirmed by gene expression analysis of diplotene spermatocytes and round spermatids obtained through fluorescence-activated cell sorting using qPCR. Our study demonstrates the fundamental role of YTHDF2 during mouse spermatogenesis and provides a potential candidate for the diagnosis of male infertility with OAT syndrome.

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