Functions And Mechanisms Of RNA Methylation Relative Proteins FTO And YTHDF2 On Mouse Spermatogonial Homeostasis

Spermatogenesis is the basis of the millions of daily produced sperm in mammalian testes,including the self-renewal of spermatogonial stem cells,the mitosis of spermatogonia,the meiosis of spermatocytes and the spermiogenesis of round spermatids.Spermatogenesis is a complicated and efficient biological process,which is tightly controlled.Dysregulation in any stage of spermatogenesis will cause male infertility.N6-methyladenosine(m~6A)is the most prevalent modification in eukaryotic mRNA.m6A plays important roles in many biological processes including cancer progression,tissue and embryo development,stem cell fate,antivirus immunology,etc.The m~6A methyltransferase METTL3 and METTL14,demethylase ALKBH5 and m~6A reader YTHDC2 are essential for mouse spermatogenesis.However,functions of other m~6A writers,erasers and readers in spermatogenesis and the underlying mechanisms of m~6A in spermatogenesis remains obescure.Here,we mainly focus on the functions and regulatory mechanisms of m~6A demethylase FTO and reader YTHDF2 in the homeostasis of mouse spermatogonia.In the present study,we used CRISPR/Cas9 to knockout Fto and Ythdc2 in cultured mouse GC-1spg cell line,detected the phenotypes,and revealed the regulatory mechanisms of FTO and YTHDF2 on phenotypes through lentivirus transduction,RNA-seq,RIP,MeRIP and RNA decay assay.1.Depletion of FTO increased the rate of giant cells in GC-1 cells.Chromosome spreading assay showed that Fto-KO induced chromosome instability(CIN).Cytometry analysis showed that the rates of aneuploidy and polyploidy were significantly increased.In the Fto-KO cells the mRNA and protein level of the core mitotic checkpoint complexes Mad3,Bub3,Bub1b and Cdc20 and the main cell cycle regulator CDK1/CCNB2 were significantly decreased,indicating that FTO induces phenotypes through downregulating the expression of the core MCC factors and the CDK1/CCNB2.2.We constructed the FTO rescue vector and the FTO mutant R313A(with no demethylase activity)vector,respectively.Wild type FTO could rescue the phenotypes and the mutant FTO could not,indicating that FTO regulates phenotypes relies on its m~6A demethylase activity.Additionally,MeRIP-qPCR results showed that Mad1,Mad2,Bub1b,Cdk1 and Ccnb2 is the direct target of FTO.RNA decay experiments showed that FTO regulates CIN and cell cycle through affecting the degradation of target mRNA.3.Depletion of YTHDF2 in spermatogonia downregulates the cell-matrix adhesion,cell spreading area and cell proliferation.Off-target analysis and rescue experiments showed that the phenotypes were not induced by off-target efficiency.RNA-seq showed that in Ythdf2-KO cells the global gene expression level was increased,and differential expressed genes(DEGs)were enriched on the GO terms cell motility and cell migration.RT-PCR results were accordant to the RNA-seq results.The downregulated genes mainly include extracellular matrix,and upregulated genes mainly include the matrix metallopeptidase(MMP)family,indicating that YTHDF2 regulates cell-matrix adhesion through the expression of matrix metallopeptidase.4.MMP3,MMP10,Adamst1 and Adamst9 from the DEGs were selected as target genes.m6A-IP-qPCR showed that the m~6A level of these four target genes was upregulated by the depletion of YTHDF2.RNA-decay results showed that the mRNA stability of MMP3,MMP10,Adamst1 and Adamst9 were affected by YTHDF2.These results suggest that YTHDF2 regulates the expression of main MMP genes through the m~6A/mRNA degradation pathway,thus affecting cell-matrix adhesion.5.Expression of Ythdf2 in mouse testes changed with age,and was highest in pubertal testes.In the adult testes,Ythdf2 mainly expressed in spermatogonia and pachytene stage spermatocytes.Spermatogenesis of Ythdf2~stra8-KOtra8-KO mouse was normal,and the histomorphology of testes and the distribution of germ cells showed no differences.The fertility of Ythdf2~stra8-KOtra8-KO mouse showed no differences with the control,indicating that YTHDF2 may be accumulated before meiosis,and was not essential for the spermiogenesis.In conclusion,the present studies reveal the function of m~6A modification in mouse cultured spermatogonia cell line,suggesting that FTO and YTHDF2 play important roles in the regulation of CIN and cell-matrix adhesion,respectively.Our findings will give theory basis for the further study of the role of RNA methylation in male fertility.