Genome-Scale Assessment of Age-Related DNA Methylation Changes in Mouse Spermatozoa

<div><p>DNA methylation plays important roles in the production and functioning of spermatozoa. Recent studies have suggested that DNA methylation patterns in spermatozoa can change with age, but the regions susceptible to age-related methylation changes remain to be fully elucidated. In this study, we conducted genome-scale DNA methylation profiling of spermatozoa obtained from C57BL/6N mice at 8 weeks (8w), 18 weeks (18w) and 17 months of age (17m). There was no substantial difference in the global DNA methylation patterns between 18w and 17m samples except for a slight increase of methylation levels in long interspersed nuclear elements in the 17m samples. We found that maternally methylated imprinting control regions (mICRs) and spermatogenesis-related gene promoters had 5–10% higher methylation levels in 8w samples than in 18w or 17m samples. Analysis of individual sequence reads suggested that these regions were fully methylated (80–100%) in a subset of 8w spermatozoa. These regions are also known to be highly methylated in a subset of postnatal spermatogonia, which might be the source of the increased DNA methylation in 8w spermatozoa. Another possible source was contamination by somatic cells. Although we carefully purified the spermatozoa, it was difficult to completely exclude the possibility of somatic cell contamination. Further studies are needed to clarify the source of the small increase in DNA methylation in the 8w samples. Overall, our findings suggest that DNA methylation patterns in mouse spermatozoa are relatively stable throughout reproductive life.</p></div

Stability of DNA methylation levels of various genomic features.

<p>Mean methylation levels (%) of CpG cytosines in promoter, exon, intron, intergenic regions LINE, LTR, SINE and L1 elements. The evolutionary ages of the L1 elements [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167127#pone.0167127.ref035" target="_blank">35</a>] are indicated. Data are shown as mean ± standard error (SE). Different letters indicate statistically significantly methylation differences (P < 0.05).</p

Identification of differentially methylated promoters.

<p>(A) Hierarchical clustering of differentially methylated promoters. Z-scored methylation levels are color-coded as shown. CGI and non-CGI promoters are also indicated. (B) Chromosome distribution of the differentially methylated promoters. (C) GO analysis of the promoters in Cluster I. Statistically significant (<i>P</i> < 0.05) GO terms are indicated with BH-corrected <i>P</i>-values. (D) Methylation levels of the promoters in Cluster I. Data are shown as mean ± SE. Different letters indicate statistically significantly methylation differences (<i>P</i> < 0.05). (E) Methylation pattern of the <i>Mael</i> promoter. The vertical axis indicates the methylation levels (%).</p

Heterogeneity of methylation levels of spermatogenesis-related promoters in 8w spermatozoa.

<p>(A) Heterogeneity of DNA methylation levels in spermatozoa. Methylation patterns of individual sequence reads are useful to verify whether subsets of spermatozoa have full methylation levels (pattern I) or not (pattern II). Note that the mean methylation levels are the same for both patterns. (B) Heterogeneity of the <i>Mael</i> promoter. <i>Mael</i> methylation patterns of 8w, 18w and 17m samples are shown. The region contains 6 CpG cytosines. The number of reads is indicated on the right side. Each line represents one read. Blue, unmethylated CpG cytosine; red, methylated CpG cytosine. (C) Analysis of individual sequence reads mapped to spermatogenesis-related gene promoters. Sequence reads were classified into five groups according to their methylation levels. The distribution of the methylation levels is shown as stacked bar charts.</p

Heterogeneity of methylation levels of mICRs in 8w spermatozoa.

<p>(A) Methylation levels of mICRs. Data are shown as mean ± SE. Different letters indicate statistically significant methylation differences (<i>P</i> < 0.05). (B) Methylation pattern of the <i>Plagl1</i> ICR. The vertical axis indicates the methylation levels (%). (C) Heterogeneity of <i>Plagl1</i> ICR. <i>Plagl1</i> ICR methylation patterns of 8w, 18w and 17m samples are shown. The region contains 9 CpG cytosines. The number of reads is indicated on the right. Blue, unmethylated CpG cytosine; red, methylated CpG cytosine. (D) Analysis of individual sequence reads. The vertical axis indicates the samples. Sequence reads were classified into five groups according to their methylation levels. The distribution of the methylation levels is shown as stacked bar charts.</p

Abnormalities in placentae derived from cloned embryos.

<p>(A) Placental weights from ICSI-derived control embryos and CB- or LatA-treated cloned embryos. Error bars indicate SD. Asterisks indicate significant difference at p < 0.05. (B) Hematoxylin and eosin staining of placentae derived from ICSI-derived control and CB- or LatA-treated cloned embryos. Abnormal distortion of the boundary between the spongiotrophoblast and labyrinth layers was observed in placentas derived from both CB- and LatA-treated cloned embryos. Bar = 500 μm.</p

Histone modifications in 1-cell stage embryos.

<p>(A-D) acH3K9, acH3K14, H3K9me2 or H3K4me2 levels in ICSI-generated and CB- or LatA-treated cloned embryos. Bar = 30 µm. (E) The intensities of immunofluorescence for acH3K9, acH3K14, H3K9me2 and H3K4me2 relative to that of H2B. They are compared with the intensities in ICSI-generated control embryos. The acetylation or methylation levels of these regions were not different between LatA- and CB-treated cloned embryos.</p

Chromosome segregation in early embryonic development.

<p>(A) Chromosomes were misaligned at metaphase and lagging chromosomes were found at anaphase in embryos ACS was occured. (B-D) Merged and bright field images of embryos with normal chromosomal segregation (NCS) and abnormal chromosomal segregation (ACS). As an example, time-lapse images of chromosome segregation at the first, second and third mitosis are shown at the 2-cell, 4-cell and 8-cell stages, respectively. Arrows indicate chromosomal fragments appearing during the division. Green, EGFP-α-tubulin; red, mRFP–H2B. Bar = 30 µm. (E) The percentages of embryos with ACS at the 2-cell, 4-cell and 8-cell stages in ICSI-generated and cloned embryos. Values with different superscripts in the same category differ significantly between ICSI-generated, CB- and LatA-treated cloned embryos.</p