As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m⁶A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m⁶A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m⁶A/mRNA degradation pathway.

Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N 6-methyladenosine (m6A), an epigenetic modification prevalent in mRNAs, is implicated in the transcriptional regulation during spermatogenesis. However, the dynamics of m6A modification in non-rodent mammalian species remains unclear. Here, we systematically investigated the profile and role of m6A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m6A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m6A pattern between porcine and murine genes with spermatogenic function. We found that m6A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m6A modification. Our data show that m6A plays the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point are three genes (SETDB1, FOXO1, and FOXO3) that are crucial to the determination of the fate of SSCs. To the best of our knowledge, this study for the first time uncovers the expression profile and role of m6A during spermatogenesis in large animals and provides insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.

The transition from gonocytes into spermatogonia takes place during the homing process. A subpopulation of undifferentiated spermatogonia in niche then shifts to spermatogonial stem cells (SSCs), accompanied by the self-renewal ability to maintain life-long fertility in males. Enormous changes in cell morphology, gene expression, and epigenetic features have been reported during spermatogenesis. However, little is known about the difference of these features in SSCs during aging. Here, we examined the dynamics of SET domain bifurcated 1 (SETDB1) expression in porcine testes. SETDB1 was expressed in postnatal undifferentiated spermatogonia, while gradually disappeared after being packed within the basal compartment of seminiferous tubules. In addition, the cell-adhesion ability, proliferative activity, and trimethylation of the histone H3 lysine 9 (H3K9me3) level were significantly altered in SETDB1-deficient porcine SSCs. Moreover, the matrix metalloproteinases 3/10 (MMP3/10) was upregulated at both mRNA and protein levels. These results illustrate the significance of SETDB1 in modulating early male germ cell development.