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. 2017 Oct;27(10):1216-1230.
doi: 10.1038/cr.2017.117. Epub 2017 Sep 15.

Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis

Zhen Lin  1 , Phillip J Hsu  2 , Xudong Xing  3 , Jianhuo Fang  3 , Zhike Lu  2 , Qin Zou  3 , Ke-Jia Zhang  1 , Xiao Zhang  4 , Yuchuan Zhou  1 , Teng Zhang  1 , Youcheng Zhang  1 , Wanlu Song  3 , Guifang Jia  4 , Xuerui Yang  3 , Chuan He  2   4 , Ming-Han Tong  1
Affiliations

Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis

Zhen Lin et al. Cell Res. 2017 Oct.

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 Vasa-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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Figures

Figure 1
Figure 1
Characterization of germ cell-specific Mettl3 mutants. (A) UPLC-MS/MS analysis of m6A percentage relative to adenosine in purified mRNA from the undifferentiated spermatogonia of controls and Mettl3-vKO mutants. Data are expressed as mean ± SD from two biological replicates. **P < 0.01, Student's t-test. (B) Gross morphology of representative testes from an adult control and age-matched Mettl3-vKO mutant. (C, D) H&E staining of control (C) and Mettl3-vKO (D) testes at age of 6 weeks. (E, F) Immunohistochemical staining for germ-cell marker GCNA (green), Sertoli cell marker SOX9 (red), and DAPI (blue) in sections of 6-week-old control (E) and Mettl3-vKO mutant (F) testes. (G, H) Whole-mount immunostaining of seminiferous tubules for GFRα1 (a marker for early stage of the undifferentiated spermatogonia, Green), PLZF (a marker for the undifferentiated spermatogonia, red), and DAPI (blue) in 4-week-old controls (G) and Mettl3-vKO mutants (H). White and yellow arrowheads indicate both GFRα1- and PLZF-positive representative As and Ap spermatogonia, respectively, in controls, whereas there are no As and even Ap spermatogonia in mutants. (Scale bar: 40 μm).
Figure 2
Figure 2
Characterization of germ cell-specific Mettl14 mutants. (A) UPLC-MS/MS analysis of m6A percentage relative to adenosine in purified mRNA from the undifferentiated spermatogonia of controls and Mettl14-vKO mutants. Data are expressed as mean ± SD from two biological replicates. **P < 0.01, Student's t-test. (B) Gross morphology of representative testes from an adult control and age-matched Mettl14-vKO mutant. (C, D) H&E staining of control (C) and Mettl14-vKO (D) testes at age of 6 weeks. (E, F) Immunohistochemical staining for the undifferentiated spermatogonia marker PLZF (red) and DAPI (blue) in sections of 6-week-old control (E) and Mettl14-vKO mutant (F) testes. (G, H) Whole-mount immunostaining of seminiferous tubules for GFRα1 (green), PLZF (red), and DAPI (blue) in 4-week-old controls (G) and Mettl14-vKO mutants (H). White and yellow arrowheads indicate both GFRα1- and PLZF-positive representative As and Ap spermatogonia, respectively, in controls (G). There is no As, even Ap spermatogonia in Mettl14-vKO mutants (H). (Scale bars: 40 μm.)
Figure 3
Figure 3
Analysis of advanced germ cell-specific Mettl3 and Mettl14 double-mutants. (A) m6A LC-MS/MS quantification in spermatid of control and different knockout mice. The data show the mean ± SD of two biological replicates, **P < 0.01, Student's t-test. (B) Morphology of testes and epididymis, from controls and Mettls-sKO double mutants. (Scale bars: 40 μm). (C) Number, total, and progressive motility of caudal epididymal sperm from 2-month-old control and Mettls-sKO double-mutant mice. Error bars represent SD, **P < 0.01, Student's t-test (n = 5-6). (D) Fluorescence staining of caudal epididymal sperms from controls and Mettls-sKO mutants with fluorescence dye-labeled peanut lectin (PNA, red) for acrosome, MitoTracker Green FM (green) for mitochondria, and DAPI (blue), respectively.
Figure 4
Figure 4
Dynamic change of m6A during spermatogenesis. (A) m6A LC–MS/MS quantification in six different developmental stages of spermatogonial cell. Un.S, undifferentiated spermatogonia; A1, type A1 spermatogonia; Prel, preleptotene spermatocytes; L/Z, lepotene/zygotene spermatocytes; Pa, Pachyotene/diplotene spermatocytes; Spd, round spermatids. The data show the mean ± SD of two biological replicates, **P < 0.01, Student's t-test. (B) Metagene distribution of m6A read density measured by m6A-seq depicting the subtranscript distribution pattern of m6A sites within the transcriptome of five different stages of spermatogenic cells. (C) RNA expression of transcripts with “emerging” or “resolving” peaks compared to unmethylated transcripts in five different stages of spermatogenic cells. A, undifferentiated spermatogonia; B, type A1 spermatogonia; C, preleptotene spermatocytes; D, pachytene/diplotene spermatocytes; E, round spermatids. “Emerging” peaks, m6A peaks with greater enrichment (enrichment ratio > 2) upon differentiation from a previous stage; “Resolving” peaks, m6A peaks with less enrichment (enrichment ratio < 0.5) upon differentiation to the next stage.
Figure 5
Figure 5
The mRNA translation dysregulations in the THY1+ SSC/progenitor cells from the Mettl3 and Mettl14 single-mutants. (A, B) Scatter plots showing the fold changes of the RPF and mRNA of the genes in the THY1+ SSC/progenitor cells upon Mettl3 (A) or Mettl14 (B) knockout. (C) Different proportions of the transcripts with m6A modification in the genes with or without differential TE in the THY1+ SSC/progenitor cells upon Mettl3 (left) or Mettl14 (right) knockout. The P-value of such difference was calculated with the Fisher's exact test. (D) Overlaps between the genes that are translationally up- (top) or down- (bottom) regulated upon Mettl3 and Mettl14 knockout. P < 0.05. (E) Heat maps showing the relative levels of the mRNA and RPF read counts of the selected genes, which are known to be involved in spermatogenesis, in the THY1+ SSC/progenitor cells from the WT, Mettl3-vKO, and Mettl14-vKO mice.
Figure 6
Figure 6
The mRNA translation dysregulations in spermatids from the Mettl3 and Mettl14 double mutants. (A) Scatter plot showing the fold changes of the RPF and mRNA of the genes in spermatids from the Mettl3 and Mettl14 double-mutant mice. (B) Different proportions of the transcripts with m6A modification in the genes with or without differential TE in spermatids upon Mettl3 and Mettl14 double mutation. The P-value of such difference was calculated with the Fisher's exact test. (C) Heat maps showing the relative levels of the mRNA and RPF read counts of the selected genes, which are known to be involved in spermatogenesis (spermatids from the WT and Mettls-sKO mice).
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