METTL16 and YTHDC1 Regulate Spermatogonial Differentiation via m6A.

Abstract

Spermatogenesis is a highly unique and intricate process, finely regulated at multiple levels, including post-transcriptional regulation. N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic mRNA, plays a significant role in transcriptional regulation during spermatogenesis. Previous research indicated extensive m6A modification at each stage of spermatogenesis, but depletion of Mettl3 and/or Mettl14 in spermatogenic cells with Stra8-Cre did not reveal any detectable abnormalities up to the stage of elongating spermatids. This suggests the involvement of other methyltransferases in the regulation of m6A modification during spermatogonial differentiation and meiosis. As a METTL3/14-independent m6A methyltransferase, METTL16 remains insufficiently studied in its roles during spermatogenesis. We report that male mice with Mettl16^vasa-cre exhibited significantly smaller testes, accompanied by a progressive loss of spermatogonia after birth. Additionally, the deletion of Mettl16 in A1 spermatogonia using Stra8-Cre results in a blockade in spermatogonial differentiation. Given YTHDC1's specific recognition for METTL16 target genes, we further investigated the role of YTHDC1 using Ythdc1-sKO mouse model. Our results indicate that Ythdc1^Stra8-cre also impairs spermatogonial differentiation, similar to the effects observed in Mettl16^Stra8-cre mice. RNA-seq and m6A-seq analyses revealed that deletion of either Mettl6 or Ythdc1 disrupted the gene expression related to chromosome organisation and segregation, ultimately leading to male infertility. Collectively, this study underscores the essential roles of the m6A writer METTL16 and its reader YTHDC1 in the differentiation of spermatogonia.