Abstract
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 V asa -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.
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Acknowledgements
M-HT was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000), the Ministry of Science and Technology of China (2014CB943101), the National Key Research and Development Program of China (2016YFC1000600), SIBS foundation, the National Natural Science Foundation of China (31471401 and 31671553), the Science and Technology Commission of Shanghai Municipality (14140901502 and 14JC1407100). This work was also supported by NIH HG008688 (CH). CH is an investigator of the Howard Hughes Medical Institute. XY was supported by the National Natural Science Foundation of China (81472855 and 91540109), the Ministry of Science and Technology of China Grant (2016YFC0906001). We thank the Histology, Flow Cytometry and Vivarium services at SIBCB, and the Genome Sequencing and High-performance Computing services from the National Protein Science Facility (Beijing) at Tsinghua University.
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Supplementary information
Supplementary information, Figure S1 (download PDF )
METTL3 and METTL14 in mouse testes. (PDF 314 kb)
Supplementary information, Figure S2 (download PDF )
Immunofluorescence detection of the methyltransferases in control and mutant testes. (PDF 195 kb)
Supplementary information, Figure S3 (download PDF )
Progressive loss of spermatogonial stem cell in Mettl3-vKO mutant mice. (PDF 300 kb)
Supplementary information, Figure S4 (download PDF )
Progressive loss of spermatogonial stem cell in Mettl3 and Mettl14 double vKO mutant mice. (PDF 192 kb)
Supplementary information, Figure S5 (download PDF )
Spermatogenesis occurs normally in Mettl3-sKO and Mettl14-sKO mutants. (PDF 641 kb)
Supplementary information, Figure S6 (download PDF )
Immunofluorescence detection of the methyltransferases in control and mutant testes.Characterization of advanced germ cell-specific Mettl3 and Mettl14 double-mutants. (PDF 793 kb)
Supplementary information, Figure S7 (download PDF )
Characterization of advanced germ cell-specific Mettl3 and Mettl14 double-mutants. (PDF 389 kb)
Supplementary information, Figure S8 (download PDF )
Combined loss of Mettl3 and Mettl14 in advanced germ cells blocked spermiogenesis. (PDF 258 kb)
Supplementary information, Figure S9 (download PDF )
Profiling of m6A in five developmental stages of spermatogenic cells. (PDF 325 kb)
Supplementary information, Figure S10 (download PDF )
Analysis of the genes with differential TE in the THY1+ SSC/progenitor cells from the Mettl3 and Mettl14 single-mutants. (PDF 143 kb)
Supplementary information, Figure S11 (download PDF )
Analysis of the genes with differential TE in the spermatocytes and spermatids from the Mettl3 and Mettl14 double-mutants. (PDF 134 kb)
Supplementary information, Figure S12 (download PDF )
Functional surveys of the translationally dysregulated genes in round spermatids from the Mettl3 and Mettl14 double-mutants. (PDF 128 kb)
Supplementary information, Figure S13 (download PDF )
Functional surveys of the translationally dysregulated genes in the spermatocytes from the Mettl3 and Mettl14 double-mutants. (PDF 310 kb)
Supplementary information, Table S1 (download XLSX )
m6A peaks in spermatogenic cells. (XLSX 6421 kb)
Supplementary information, Table S2 (download XLSX )
GO analyses of the methylated transcripts. (XLSX 150 kb)
Supplementary information, Table S3 (download XLSX )
The methylated transcripts of genes essential for spermatogenic cell development. (XLSX 144 kb)
Supplementary information, Table S4 (download XLSX )
Altered TE in Mettl3 and Mettl14 single-mutant SSCs/progenitor cells. (XLSX 1562 kb)
Supplementary information, Table S5 (download XLSX )
“Emerging” and “resolving” m6A peaks in pachytene/diplotene spermatocytes. (XLSX 16 kb)
Supplementary information, Table S6 (download XLSX )
Altered TE in Mettl3 and Mettl14 double-mutant spermatids. (XLSX 1008 kb)
Supplementary information, Table S7 (download XLSX )
Altered TE in Mettl3 and Mettl14 double-mutant spermatocytes. (XLSX 1156 kb)
Supplementary information, Table S8 (download PDF )
The primers used for mouse genotyping (PDF 86 kb)
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Lin, Z., Hsu, P., Xing, X. et al. Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis. Cell Res 27, 1216–1230 (2017). https://doi.org/10.1038/cr.2017.117
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DOI: https://doi.org/10.1038/cr.2017.117
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