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

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Heterogeneity of methylation levels of spermatogenesis-related promoters in 8w spermatozoa.(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 Mael promoter. Mael 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.
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pone.0167127.g002: Heterogeneity of methylation levels of spermatogenesis-related promoters in 8w spermatozoa.(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 Mael promoter. Mael 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.

Mentions: We next focused on the methylation patterns of individual sequence reads, which are useful to determine whether a subset of spermatozoa have full methylation levels (pattern I in Fig 2A) or not (pattern II in Fig 2A). We used only reads containing at least 5 CpG cytosines and analyzed differentially methylated promoters where ≥20 reads were mapped. Seven spermatogenesis-related gene promoters were successfully analyzed. We found that 5–15% of reads in these promoters were >80% methylated and most of the other reads were <20% methylated (Fig 2B and 2C), supporting pattern I in Fig 2A. Although we carefully purified the spermatozoa, it was difficult to completely exclude the possibility of somatic cell contamination. As spermatogenesis-related promoters are frequently hypermethylated in somatic cells, the higher methylation levels of these regions in 8w samples could be attributable to somatic cell contamination. If so, the highly methylated reads must be excluded from the analysis. Considering that ≥60% methylated reads were specifically observed in 8w samples (Fig 2C), we recalculated the methylation levels of the spermatogenesis-related promoters without these ≥60% methylated reads. After the removal of the ≥60% methylated reads, most of these regions had similar methylation levels in all samples (S5A Fig). This result suggested that if our spermatozoa samples were contaminated with somatic cells, spermatogenesis-related promoters might not be differentially methylated among 8w, 18w and 17m spermatozoa.


Genome-Scale Assessment of Age-Related DNA Methylation Changes in Mouse Spermatozoa
Heterogeneity of methylation levels of spermatogenesis-related promoters in 8w spermatozoa.(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 Mael promoter. Mael 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.
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pone.0167127.g002: Heterogeneity of methylation levels of spermatogenesis-related promoters in 8w spermatozoa.(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 Mael promoter. Mael 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.
Mentions: We next focused on the methylation patterns of individual sequence reads, which are useful to determine whether a subset of spermatozoa have full methylation levels (pattern I in Fig 2A) or not (pattern II in Fig 2A). We used only reads containing at least 5 CpG cytosines and analyzed differentially methylated promoters where ≥20 reads were mapped. Seven spermatogenesis-related gene promoters were successfully analyzed. We found that 5–15% of reads in these promoters were >80% methylated and most of the other reads were <20% methylated (Fig 2B and 2C), supporting pattern I in Fig 2A. Although we carefully purified the spermatozoa, it was difficult to completely exclude the possibility of somatic cell contamination. As spermatogenesis-related promoters are frequently hypermethylated in somatic cells, the higher methylation levels of these regions in 8w samples could be attributable to somatic cell contamination. If so, the highly methylated reads must be excluded from the analysis. Considering that ≥60% methylated reads were specifically observed in 8w samples (Fig 2C), we recalculated the methylation levels of the spermatogenesis-related promoters without these ≥60% methylated reads. After the removal of the ≥60% methylated reads, most of these regions had similar methylation levels in all samples (S5A Fig). This result suggested that if our spermatozoa samples were contaminated with somatic cells, spermatogenesis-related promoters might not be differentially methylated among 8w, 18w and 17m spermatozoa.

View Article: PubMed Central - PubMed

ABSTRACT

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&ndash;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&ndash;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.

No MeSH data available.