Matsubayashietal_2017_JEB_AFLP_alldata

AFLP genotype data for all individuals used in the paper

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

Additional file 5: Fig. S1. of Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer

Validation of placental miRNAs expression using qRT-PCR. The boxplots show the expression levels of miR-197-5p (a), miR-4697-5p (b), miR-4721 (c), miR-5006-5p (d), miR-575 (e), miR-6893-5p (f), miR-125a-5p (g), miR-1260b (h), miR-224-3p (i), miR-331-3p (j), miR-365a-3p (k), miR-495-3p (l), miR-518b (m), miR-518f-3p (n), miR-543 (o) and miR-7977 (p). Data were normalized to RNU44 expression and are presented as boxplots with whiskers. The upper and lower limits of the boxes present the 75th and 25th percentiles, respectively. The upper and lower whiskers represent the maximum and minimum values that are no more than 1.5 times the span of the interquartile range (range of the values between the 25th and the 75th percentiles). The circles indicate the outliers. The median is indicated by the line in each box. They were analyzed using the Steel-Dwass test and considered statistically significant when p < 0.05. (PDF 1684 kb

Additional file 1: Table S1. of Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer

Patients’ information. Blastocysts were graded using a blastocyst scoring system [46]. FSH follicular stimulating hormone, HMG human menopausal gonadotropin. (XLS 51 kb

Additional file 2: Table S2. of Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer

List of TaqMan probe ID numbers. (XLS 29 kb

Additional file 9: Table S7. of Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer

KEGG enrichment analysis of target genes. (XLS 45 kb