Genome-wide DNA methylation changes in human spermatogenesis

Abstract

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

Keywords: DNA methylation; epigenetics; germline; human spermatogenesis; infertility; male germ cells; methylome; transposable elements.

Graphical abstract

Figure 1

Primary spermatocytes exhibit genome-wide reduced DNA methylation levels (A) Schematic illustration on the retrieval of whole-genome methylome data from germ cells of samples with normal spermatogenesis (control, CTR). CpGs refer to the mean CpG number captured by enzymatic methyl-sequencing in all germ cell fractions (n = 12). (B) Line plot depicts the methylation in 50 imprinting control regions (ICRs) for each CTR sample and germ cell type compared to published blood and sperm samples.. (C) Boxplots display the mean global DNA methylation levels. Statistical tests: ANOVA test followed by Tukey-HSD-test: ^∗∗∗ <0.001 of 4C compared to all other germ cells. Data are represented as median (center line), upper/lower quartiles (box limits), 1.5× interquartile range (whiskers). (D) Line plot shows mean methylation levels per group across gene bodies divided into 50 intervals (bins) and 5 kb upstream and downstream of the transcriptional start sites (TSSs) and transcriptional end sites (TESs). (E) Violin plots represent methylated CpGs across different genomic compartments. Undiff, undifferentiated spermatogonia; Diff, differentiating spermatogonia; 4C, primary spermatocytes; 1C, spermatids/sperm. (A) was created with BioRender.com. See also Figure S1.

Figure 2

Differentially methylated regions during spermatogenesis are enriched at SINE repeats (A) Heatmaps display methylation values and CpG numbers of the differentially methylated regions (DMRs) of all germ cell type comparisons. (B) DMRs are associated with genes and promoters. (C) Frequency of CpG number per DMR within the different group comparisons. (D) Violin plot depicts distribution of the DMR width in each group comparison. (E) Overlap (100%) of the DMRs among the different group comparisons. The exclusive DMRs are indicated by a dot, and the overlap of DMRs is shown by connecting nodes. (F) Distribution of DMRs per chromosome scaled for chromosomal size (base pairs) and normalized by their total count within one group. (G) Enrichment of DMRs for general genomic features and genomic repeats. Positive and negative enrichments are indicated by Z score. Displayed annotations, p < 0.00019 by permutation tests. Color coding of the group comparisons are depicted in (A). Undiff, undifferentiated spermatogonia; Diff, differentiating spermatogonia; 4C, primary spermatocytes; 1C, spermatids/sperm.

Figure 3

Hypomethylated regions in spermatids/sperm are enriched for specific TF binding sites (A) Depicted are the enriched sequences of known motifs identified by HOMER. HOMER analysis was run for all DMR comparisons and significant results are displayed (p value <0.01, false discovery rate (FDR) < 0.05). Undiff, undifferentiated spermatogonia; Diff, differentiating spermatogonia; 4C, primary spermatocytes; 1C, spermatids/sperm. (B) Dot plots show the average single-cell expression of the top three transcription factors (TFs) with enriched motifs among the DMRs identified with HOMER. SPC, spermatocytes; SPD, spermatids.

Figure 4

Hypomethylated regions in the spermatids/sperm methylomes mark spermatid-specific genes (A) Single-cell expression of hypomethylated DMR-associated genes with specific expression in spermatids and hypermethylated DMR-associated genes with specific expression in undifferentiated spermatogonia of the undifferentiated spermatogonia vs. 1C DMRs. (B) Example of the DMR methylation within the AGPAT3 locus that is hypermethylated in Undiff and Diff and hypomethylated in 4C and 1C and specifically expressed in SPD. H3K36me3 histone modification data (GSE40195) in human sperm is also shown. Undiff, undifferentiated spermatogonia; Diff, differentiating spermatogonia; 4C, primary spermatocytes; 1C, spermatids/sperm; SPC, spermatocytes; SPD, spermatids. See also Figure S3.

Figure 5

Disturbed spermatogenesis displays methylome changes at TEs and spermatogenesis genes (A) Schematic illustration on the retrieval of whole-genome methylome data of germ cells from samples with disturbed spermatogenesis (CZ, cryptozoospermia). (B) Boxplots show the proportion of cell types among the sorted cells in the CTR and CZ subjects. Data are represented as median (center line), upper/lower quartiles (box limits), 1.5× interquartile range (whiskers). (C) Heatmaps display methylation values of the differentially methylated regions (DMRs) between CTR and CZ of the same cell type (Undiff vs. Undiff, Diff vs. Diff, and 4C vs. 4C). (D) Distribution of the CTR/CZ DMRs per chromosome scaled for chromosomal size (base pairs) and normalized by their total count within one group. (E) Enrichment of CTR/CZ DMRs for functional general genomic regions and genomic repeats. Positive and negative enrichments are indicated by Z score. Displayed annotations, p < 0.00019 by permutation tests. (F) Violin plots showing the CpG methylation of evolutionary younger (white boxes: L1Hs, L1PA2-5, and SVA_D/F) and older (gray boxes: HERVH-int and L1M7) TEs in CTR and CZ germ cells. Color coding of the group comparisons are depicted in (B). Undiff, undifferentiated spermatogonia; Diff, differentiating spermatogonia; 4C, primary spermatocytes; 1C, spermatids/sperm. (A) created with BioRender.com. See also Figures S4 and S5.