Comprehensive Analysis of the Transcriptome-Wide m6A Methylome in Shaziling Pig Testicular Development

Abstract

RNA N⁶-methyladenosine (m⁶A) modification is one of the principal post-transcriptional modifications and plays a dynamic role in testicular development and spermatogenesis. However, the role of m⁶A in porcine testis is understudied. Here, we performed a comprehensive analysis of the m⁶A transcriptome-wide profile in Shaziling pig testes at birth, puberty, and maturity. We analyzed the total transcriptome m⁶A profile and found that the m⁶A patterns were highly distinct in terms of the modification of the transcriptomes during porcine testis development. We found that key m⁶A methylated genes (AURKC, OVOL, SOX8, ACVR2A, and SPATA46) were highly enriched during spermatogenesis and identified in spermatogenesis-related KEGG pathways, including Wnt, cAMP, mTOR, AMPK, PI3K-Akt, and spliceosome. Our findings indicated that m⁶A methylations are involved in the complex yet well-organized post-transcriptional regulation of porcine testicular development and spermatogenesis. We found that the m⁶A eraser ALKBH5 negatively regulated the proliferation of immature porcine Sertoli cells. Furthermore, we proposed a novel mechanism of m⁶A modification during testicular development: ALKBH5 regulated the RNA methylation level and gene expression of SOX9 mRNA. In addition to serving as a potential target for improving boar reproduction, our findings contributed to the further understanding of the regulation of m⁶A modifications in male reproduction.

Keywords: ALKBH5; SOX9; Shaziling pig; mRNA m6A methylation; testis.

Figure 1

Global m⁶A levels and overview of m⁶A methylation profiles in porcine testes. (A) m⁶A dot blot detection of the global m⁶A modification levels in 1-day-old (D1), 75-days-old (D75), and 150-days-old (D150) Shaziling boar testes (n = 3). (B) Metagene plots demonstrating the regions of m⁶A peaks identified throughout the transcripts genome-wide in the D1, D75, and D150 groups. (C) Pie charts illustrating the distribution of m⁶A peaks in mRNA gene structures. (D) Top motifs enriched with m⁶A peaks in the D1, D75, and D150 groups.

Figure 2

Transcriptome-wide m⁶A analysis in porcine testes. (A) The number of shared and unique m⁶A peaks in three groups. (B) The Venn diagram shows the number of m⁶A-related genes in three groups. (C) Volcano plots showing the significantly differential m⁶A peaks compared between the D75 and D1 groups and (D) between the D150 and D75 groups.

Figure 2

Transcriptome-wide m⁶A analysis in porcine testes. (A) The number of shared and unique m⁶A peaks in three groups. (B) The Venn diagram shows the number of m⁶A-related genes in three groups. (C) Volcano plots showing the significantly differential m⁶A peaks compared between the D75 and D1 groups and (D) between the D150 and D75 groups.

Figure 3

Conjoint analysis of m⁶A-Seq and RNA-Seq data in porcine testes. (A) Four quadrant plots showing differentially expressed genes with differentially methylated m⁶A peaks (|log2.fc| ≥ 1, p < 0.05) among the studied groups. (B) The RT-qPCR analysis of five genes (BAG6, SOX9, KDM3A, PRM2, and RARA) and (C) core m⁶A methylation-related genes in three groups (D1, D75, and D150) determined the relative mRNA levels. *, ** and *** represent p < 0.05, p < 0.01, and p < 0.001 respectively. (D) GO enrichment analysis of Diff_1 gene set in D75 vs. D1. (E) KEGG pathway enrichment analysis of Diff_1 gene set in D75 vs. D1.

Figure 4

The biological profile and potential mechanisms of ALKBH5 in iSCs. (A) The immunohistochemical (IHC) analysis of ALKBH5 expressions in the testes of Shaziling pigs at different ages (D1, D75, and D150) and cell types are pointed out by arrows of various colors (n = 3). Black arrow, Leydig cells; blue arrow, Sertoli cells; pink arrow, spermatogonia; green arrow, spermatocytes; red arrow, spermatids; (B) Immunofluorescence (IF) analysis showed colocalization of ALKBH5 expression with SOX9 (a marker of Sertoli cells) in Shaziling pig testes at D150 (n = 3). (C) Western blot analysis detected the expression of ALKBH5 after silencing. β-actin was used as an internal control. (D) CCK-8 assay estimated the effects of ALKBH5 silencing on cell viability in iSCs. (E) Annexin-V/PI double staining assay evaluated the cell apoptosis rates after ALKBH5 silencing. (F) Western blot analysis detected the expression of BAX and Caspase-3 after ALKBH5 silencing. β-actin was used as an internal control. *, ** and *** represent p < 0.05, p < 0.01, and p < 0.001 respectively.

Figure 4

The biological profile and potential mechanisms of ALKBH5 in iSCs. (A) The immunohistochemical (IHC) analysis of ALKBH5 expressions in the testes of Shaziling pigs at different ages (D1, D75, and D150) and cell types are pointed out by arrows of various colors (n = 3). Black arrow, Leydig cells; blue arrow, Sertoli cells; pink arrow, spermatogonia; green arrow, spermatocytes; red arrow, spermatids; (B) Immunofluorescence (IF) analysis showed colocalization of ALKBH5 expression with SOX9 (a marker of Sertoli cells) in Shaziling pig testes at D150 (n = 3). (C) Western blot analysis detected the expression of ALKBH5 after silencing. β-actin was used as an internal control. (D) CCK-8 assay estimated the effects of ALKBH5 silencing on cell viability in iSCs. (E) Annexin-V/PI double staining assay evaluated the cell apoptosis rates after ALKBH5 silencing. (F) Western blot analysis detected the expression of BAX and Caspase-3 after ALKBH5 silencing. β-actin was used as an internal control. *, ** and *** represent p < 0.05, p < 0.01, and p < 0.001 respectively.

Figure 5

ALKBH5 regulated the level of RNA methylation and gene expression of SOX9 mRNA in vitro. (A) Total m⁶A levels were measured in iSC-extracted mRNAs after ALKBH5 silencing. (B) Motif analysis using the HOMER program identified “AAACC” as the m⁶A consensus motif in SOX9 3′UTR and highlighted by yellow. The red box indicated the m⁶A peak. (C) ALKBH5 silencing significantly up-regulated the mRNA level of SOX9. (D) MeRIP-qPCR analysis confirmed that ALKBH5 silencing significantly increased the mRNA m⁶A level of SOX9. (E) Dual-luciferase assays indicated that the relative fluorescence activities of the SOX9-MUT group (mutated m⁶A motif) were significantly lower than those of the SOX9-WT group in ALKBH5 silencing 293T cells. * p  <  0.05, ** p  <  0.01, *** p  <  0.001.