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. 2023 Aug;21(4):729-741.
doi: 10.1016/j.gpb.2021.08.006. Epub 2021 Sep 17.

Transcriptome-wide Dynamics of m6A mRNA Methylation During Porcine Spermatogenesis

Zidong Liu  1 , Xiaoxu Chen  1 , Pengfei Zhang  1 , Fuyuan Li  1 , Lingkai Zhang  1 , Xueliang Li  1 , Tao Huang  1 , Yi Zheng  1 , Taiyong Yu  1 , Tao Zhang  2 , Wenxian Zeng  3 , Hongzhao Lu  4 , Yinghua Lv  5
Affiliations

Transcriptome-wide Dynamics of m6A mRNA Methylation During Porcine Spermatogenesis

Zidong Liu et al. Genomics Proteomics Bioinformatics. 2023 Aug.

Abstract

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. N6-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.

Keywords: N(6)-methyladenosine; Pig; SETDB1; Spermatogenesis; Spermatogonial stem cell.

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Conflict of interest statement

The authors have declared no competing interests.

Figures

Figure 1
Figure 1
Identification of porcine male germ cells A. Immunocytochemistry showing the expression of UCHL1 in the SPG. B.  Immunocytochemistry showing the expression of SYCP3 in the freshly isolated PS. C. Immunocytochemistry showing the expression of CD63 in the RS. SPG, spermatogonium; PS, pachytene spermatocyte; RS, round spermatid; UCHL1, ubiquitin carboxyl-terminal esterase L1; SYCP3, synaptonemal complex protein 3; CD63, CD63 Molecule; DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 100 µm.
Figure 2
Figure 2
Distribution pattern of m6A peaks along transcripts A. LC-MS/MS analysis of m6A percentage relative to adenosine in SPG, PS, and RS. B. m6A peak distribution within different gene contexts: startC, CDS, stopC, 3′ UTR, and 5′ UTR. C. Accumulation of m6A-IP reads along transcripts in SPG, PS, and RS. Each transcript is divided into three parts: 5′ UTR, CDS, and 3′ UTR. D.–F. The top 3 used motifs among m6A peaks in SPG (D), PS (E), and RS (F). LC-MS/MS, liquid chromatography-tandem mass spectrometry; startC, start codon; CDS, coding sequence; stopC, stop codon; UTR, untranslated region.
Figure 3
Figure 3
m6A modification pattern during porcine spermatogenesis A. Venn diagram showing the pattern of m6A-modified genes in SPG, PS, and RS. B. The enriched biological processes of continuously methylated genes during spermatogenesis by GO analysis. C. The enriched biological processes of DMGs between PS and SPG by GO analysis. D. The enriched biological processes of DMGs between RS and PS by GO analysis. Each dot plot shows gene ratio values of the top 10 significant enrichment terms. E. Venn diagram showing the overlapping m6A-modified genes between murine and porcine in SPG, PS, and RS. F. Different proportions of the overlapping methylated genes related or unrelated to spermatogenesis in murine and porcine in SPG, PS, and RS. The P value of such difference was calculated with the Chi-square test. GO, Gene Ontology; DMG, differentially methylated gene.
Figure 4
Figure 4
Gene expression pattern during porcine spermatogenesis A. The heatamap of DEGs between PS and SPG. B. The heatamap of DEGs between RS and PS. C. The enriched biological processes of DEGs between PS and SPG by GO analysis. D. The enriched biological processes of DEGs between RS and PS by GO analysis. Each dot plot shows gene ratio values of the top 10 significant enrichment terms. DEG, differentially expressed gene.
Figure 5
Figure 5
m6A-regulatedgene expression during porcine spermatogenesis A. The m6A modification distribution within stage-specific gene contexts. Number in the red part represents the number of genes specifically methylated in SPG, PS, or RS. B. and C. The number of up- or down-regulated genes during porcine spermatogenesis stratified by up-methylated (B) or down-methylated (C) genes. The P value of such difference was calculated with the Chi-square test. D. The enriched biological processes of m6A positively regulated genes between PS and SPG by GO analysis. E. The enriched biological processes of m6A positively regulated genes between RS and PS by GO analysis. Each dot plot shows gene ratio values of the top 10 significant enrichment terms.
Figure 6
Figure 6
Knockdown of METTL3 in porcineSSCs induced the abnormal gene expression A. Distribution of m6A on SETDB1 and FOXO3 during porcine spermatogenesis. Blue and red bars indicate the input and IP read coverage, respectively. B. Bar chart showing the m6A levels of SETDB1 and FOXO3 in SPG, PS, and RS validated by m6A-RIP-qPCR. C. Bar chart showing the mRNA levels of SETDB1 and FOXO3 in porcine SPG, PS, and RS validated by qRT-PCR. D. Immunocytochemistry showing the expression of UCHL1 in porcine SSCs. E. m6A dot blot analysis of the METTL3 knockdown (siMETTL3). siCtrl was used as a negative control. Methylene blue staining was used to evaluate RNA amount. F. Representative images of EdU incorporation in the cells transfected with siMETTL3 or siCtrl. The quantification analysis of EdU incorporation in the cells transfected with siMETTL3 or siCtrl was shown on the right. G. Bar chart showing the m6A levels of the targeted genes in the cells transfected with siMETTL3 or siCtrl validated by m6A-RIP-qPCR. H. Bar chart showing the relative expression level of METTL3, SETDB1, FOXO1, and FOXO3 in the cells transfected with siMETTL3 or siCtrl detected by qRT-PCR. Data are presented as mean ± SEM. The P value was calculated with one-way ANOVA analysis followed by Bonferroni multiple-comparison test and unpaired t-test, *, P < 0.05; **, P < 0.01; ns, no significance. SSC, spermatogonial stem cell; IP, immunoprecipitation; EdU, 5-ethynyl-2′-deoxyuridine; SEM, standard error of mean. Scale bar, 100 µm.
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