Figure 3

Analysis of sperm DNA methylation according to the cryoprotectant. (a) Principal Component Analysis showing the distribution of the fresh and cryopreserved sperm samples according to the DNA methylation ratio of each cytosine within each individual sample (n = 12 sperm samples per condition). No clear segregation was observed among the different treatments. (b) Frequency plot showing the number of cytosines in CpG sites according to their methylation ratio (0–1) for each condition (fresh and cryopreserved with DMSO, MeOH and glycerol). The x axis shows 20 classes of 0.05 interval values (methylation ratios ranging from 0–0.05 to 0.95–1.0). The percentages written above the classes 0–0.05 and 0.95–1.0 indicate the percentage of the total cytosines in CpG sites in these 2 classes. (c) Histogram showing the distribution of the number of paired cytosines according to the number of males sharing them, in each comparison between fresh and cryopreservation with a given cryoprotectant. Results are expressed as a percentage of the total number of paired cytosines (fresh vs DMSO: 748,770; fresh vs MeOH: 1,645,797; fresh vs glycerol: 714,554). (d) Venn diagram of differentially methylated cytosines (DMCs) obtained in each comparison between fresh and each cryoprotectant. The numbers indicate the number of DMCs in each group. e. Histogram showing the distribution of the different classes of DMCs in each comparison: hypomethylated (> 50% of males having this DMC as hypomethylated), hypermethylated (> 50% of males having this DMC as hypermethylated) and unclassified methylation status (DMCs whose methylation status between males was too variable to be included into one of the first two classes). Values are expressed as a percentage of total DMCs: (335 in DMSO, 564 in MeOH and 357 in glycerol).