Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 13;15(4):968-982.
doi: 10.1016/j.stemcr.2020.09.001.

WTAP Function in Sertoli Cells Is Essential for Sustaining the Spermatogonial Stem Cell Niche

Gong-Xue Jia  1 , Zhen Lin  2 , Rong-Ge Yan  3 , Guo-Wen Wang  3 , Xiao-Na Zhang  3 , Cen Li  4 , Ming-Han Tong  5 , Qi-En Yang  6
Affiliations

WTAP Function in Sertoli Cells Is Essential for Sustaining the Spermatogonial Stem Cell Niche

Gong-Xue Jia et al. Stem Cell Reports. .

Abstract

Sertoli cells are the major component of the spermatogonial stem cell (SSC) niche; however, regulatory mechanisms in Sertoli cells that dictate SSC fate decisions remain largely unknown. Here we revealed features of the N6-methyladenosine (m6A) mRNA modification in Sertoli cells and demonstrated the functions of WTAP, the key subunit of the m6A methyltransferase complex in spermatogenesis. m6A-sequencing analysis identified 21,909 m6A sites from 15,365 putative m6A-enriched transcripts within 6,122 genes, including many Sertoli cell-specific genes. Conditional deletion of Wtap in Sertoli cells resulted in sterility and the progressive loss of the SSC population. RNA sequencing and ribosome nascent-chain complex-bound mRNA sequencing analyses suggested that alternative splicing events of transcripts encoding SSC niche factors were sharply altered and translation of these transcripts were severely dysregulated by Wtap deletion. Collectively, this study uncovers a novel regulatory mechanism of the SSC niche and provide insights into molecular interactions between stem cells and their cognate niches in mammals.

Keywords: Sertoli cell; WTAP; alternative splicing; m(6)A; niche; spermatogonial differentiation; spermatogonial stem cell.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
Function Analysis of m6A in Mouse Sertoli Cells (A) Immunostaining for METTL3 and GATA4, METTL14 and GATA4, or WTAP and SOX9 with DAPI in mouse testes at P6. The arrows indicate Sertoli cells. Scale bars represent 40 μm. (B) Average m6A sites per gene in distinct RNA sequence regions including 3′ UTR, CDS, and 5′ UTR. (C and D) Average fold enrichment over input for m6A peaks of selected genes specially expressed in Sertoli cells (C) or related to spermatogenesis (D). Two independent Sertoli cell samples isolated from 110 mice were used (n = 2). (E–G) Pie chart of molecular types (E), phenotype association analysis (F), and biological process GO analysis (G) of m6A-enriched genes in Sertoli cells. The bubble size indicates the number of matched genes. See also Figure S1, Tables S1, S2, and S3.
Figure 2
Figure 2
Progressive Loss of Undifferentiated Spermatogonia Caused by Wtap Deletion in Sertoli Cells (A) Immunostaining for GATA4 and m6A or WTAP and SOX9 in testis cross sections from the indicated genotypes at P6. The arrows indicate Sertoli cells. Scale bars represent 20 μm. (B–E) Testes (B) of control and Wtap-sKO male mice at P180. Scale bar represents 2 mm. Testis/body weight ratios at P6, P14, P21, P28, P35, P60, P120, and P180 (C), litter sizes from P60 to P150 (D), and sperm counts of cauda epididymis at P180 (E) from the indicated genotypes. At least three mice were used for each time point (n = 3 different animals). Ten control or Wtap-sKO animals were used in fertility test (n = 10 different animals). ∗∗p < 0.01 and ∗∗∗p < 0.001. (F–J) H&E staining of seminiferous tubules (F) from control and Wtap-sKO males at P6, P35, P60, and P180. Asterisks indicate SCO tubules. H&E staining and SOX9 and GCNA1 immunostaining (G) of SCO tubules from Wtap-sKO males. Quantification of seminiferous tubules from Wtap-sKO testes containing degenerated spermatogenesis and SCO phenotype (H) at P35, P60, P120, and P180. Immunostaining of LIN28A and SOX9 (I) in control and Wtap-sKO testes at P6, P35, P60, and P180. Statistics of the number of LIN28A+ cells per SOX9+ cell (J) from the indicated genotypes at P6, P21, P35, P60, and P180. At least 1500 SOX9+ cells were counted per genotype at each time point. Scale bars represent 40 μm. Three animals were used for control or Wtap-sKO at each time point (n = 3 different animals). ∗∗p < 0.01 and ∗∗∗p < 0.001. See also Figures S2–S4.
Figure 3
Figure 3
Impaired SSC Self-renewal and Proliferation Caused by Wtap Deletion in Mouse Sertoli Cells (A–D) Whole-mount immunostaining of GFRA1 and FOXO1 (A) and TUNEL and LIN28A (B) in control and Wtap-sKO seminiferous tubules at P21. Whole-mount immunostaining of EdU and LIN28A (C) in control and Wtap-sKO seminiferous tubules after 2 h or 7 days post EdU injection at P14. Quantifications of EdU+ As, Apr, and Aal spermatogonia in Lin28A+ cells (D) of control and Wtap-sKO testes. At least 900 LIN28A+ cells were counted per genotype for each time point. Three animals were used for control or Wtap-sKO at each time point (n = 3 different animals). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bars represent 40 μm. (E–G) The schematic diagram (E) of germ cell transplantation experiment. Quantification of THY1+ cells obtained using MACS (F) and SSC numbers in THY1+ undifferentiated spermatogonia (G) from control and Wtap-sKO males. SSC numbers were derived from donor-derived colonies of spermatogenesis in recipient testes and normalized to 105 cells injected. THY1+ cells from three control or Wtap-sKO animals (4 weeks old) were isolated and transplanted into testes of six recipients (n = 3 different animals). ∗∗p < 0.01. (H) qRT-PCR analysis of Thy1, Cxcr4, Gfra1, Id4, Lin28, Zbtb16, and Rb1 transcript abundances in THY1+ cells from control and Wtap-sKO testes. Four control or Wtap-sKO animals were used (n = 4 different animals). ∗p < 0.05. See also Figures S4 and S5.
Figure 4
Figure 4
Transcriptional and Translational Dysregulations in Sertoli Cells from Wtap-sKO Mice (A and B) Heat map showing the DEGs (A) and DTGs (B) between control and Wtap-sKO animals and their function enrichment. Gene differences with |log2fold change| ≥ 1 and p < 0.01 are identified as significant. The enriched biological process GO terms in downregulated (blue boxes) and upregulated (red boxes) genes upon Wtap knockout are shown in the right panels. (C) Scatterplots showing the relationship of fold changes between mRNA FPKM and RPF TE of genes in Sertoli cells upon Wtap knockout. Red, blue, green, and gray plots indicate genes belonging to both DEGs and DTGs, DEGs only, DTGs only, and neither DEGs nor DTGs. (D) Heat maps showing the relative levels of mRNA FPKM and RPF TE of genes involving SSC maintenance and spermatogonial differentiation in Sertoli cells. (E) Interaction network showing genes involved in spermatogenesis regulation. Two genes with regulatory relationships are connected directly, DEGs and DTGs are marked in blue and green respectively. All analyses were performed on three different control or Wtap-sKO samples (n = 3 different biological replicates). ∗p < 0.05. See also Table S4.
Figure 5
Figure 5
Effects of WTAP-Mediated m6A Modification on Transcriptional and Post-transcriptional Regulation of Sertoli Cell Gene Expression (A–D) Venn diagram showing numbers of DEGs and DTGs with or without m6A modification (A), proportions of m6A-enriched genes on differential transcriptional and translational changes (B), mRNA FPKM of DEGs and RPF TE of DTGs with or without m6A modification (C), and heat maps showing relative levels of mRNA FPKM and RPF TE of the main m6A regulators (D) in control and Wtap-sKO Sertoli cells. (E–I) Statistics of five types of differential AS events (E) on mRNA transcripts in control and Wtap-sKO Sertoli cells. Venn diagram (F) showing numbers of DEGs and DTGs with or without differential AS events in Sertoli cells. Average m6A sites (G) of genes with or without differential AS events. Average inclusion levels (H) and cumulative frequency curves for inclusion levels (I) of A5SS and A3SS, MXE, RI, and SE in genes with or without m6A modification between control and Wtap-sKO animals. (J) Interaction network showing genes involved in spermatogenesis regulation. Two genes with regulatory relationships are connected directly, genes with m6A modification and differential AS events are marked in red and yellow respectively. All analyses were performed on three different control or Wtap-sKO samples (n = 3 different biological replicates). ∗p < 0.05. See also Table S5.
Figure 6
Figure 6
Decreased Expression of SSC Niche Factors Caused by Wtap Deletion (A) mRNA and RNC mRNA reads of Cxcl12 and Csf1 in control and Wtap-sKO samples. The m6A sites are marked as red triangles. (B) qRT-PCR analysis of Wtap, Cxcl12, Ar, Csf1, Etv5, Wt1, Gata4, Fgf2, Bmp4, Cyp26b1, and Gdnf transcript abundances in control and Wtap-sKO Sertoli cells. (C and D) Western blot (C) and quantitative data (D) showing CXCL12, GDNF, CSF1, and CYP26B1 expression in control and Wtap-sKO testes. All quantitative data are presented as the mean ± SEM for n = 3 independent biological replicates. ∗p < 0.05 and ∗∗p < 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adhikari S., Xiao W., Zhao Y.L., Yang Y.G. m6A: signaling for mRNA splicing. RNA Biol. 2016;13:756–759. - PMC - PubMed
    1. Balacco D.L., Soller M. The m6A writer: rise of a machine for growing tasks. Biochemistry. 2019;58:363–378. - PubMed
    1. Basciani S., De Luca G., Dolci S., Brama M., Arizzi M., Mariani S., Rosano G., Spera G., Gnessi L. Platelet-derived growth factor receptor β-subtype regulates proliferation and migration of gonocytes. Endocrinology. 2008;149:6226–6235. - PubMed
    1. Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. - PMC - PubMed
    1. Chen M., Zhang L.J., Cui X.H., Lin X.W., Li Y.Q., Wang Y.Q., Wang Y.B., Qin Y., Chen D.H., Han C.S. Wt1 directs the lineage specification of Sertoli and granulosa cells by repressing Sf1 expression. Development. 2017;144:44–53. - PubMed

Publication types

MeSH terms

LinkOut - more resources