Embryonic imprinting perturbations do not originate from superovulation-induced defects in DNA methylation acquisition  Michelle M. Denomme, B.Sc., Liyue Zhang, B.Sc., Mellissa R.W. Mann, Ph.D.  Fertility and Sterility  Volume 96, Issue 3, Pages 734-738.e2 (September 2011) DOI: 10.1016/j.fertnstert.2011.06.055 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Methylation analysis of the Snrpn ICR (A), Kcnq1ot1 ICR (B), Peg3 DMR (C), and H19 ICR (D) in individual oocytes derived from spontaneously ovulating B6(CAST7p6)XB6 females. For each gene, n = 10. Black circles indicate methylated CpGs. White circles indicate unmethylated CpGs. U = untreated; C = CAST allele; B = B6 allele. Each row represents an individual oocyte; designation indicated to the left. The Snrpn ICR region analyzed contains 16 CpGs; a polymorphism eliminates CpG dinucleotide 1 in the CAST allele. The Kcnq1ot1 ICR region analyzed contains 20 CpGs. The Peg3 DMR region analyzed contains 24 CpGs; a polymorphism eliminates CpG dinucleotide 22 in the B6 allele. The H19 ICR region analyzed contains 17 CpGs; a polymorphism eliminates CpG dinucleotide 8 in the B6 allele. Fertility and Sterility 2011 96, 734-738.e2DOI: (10.1016/j.fertnstert.2011.06.055) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Methylation analysis of the Snrpn ICR (A), Kcnq1ot1 ICR (B), Peg3 DMR (C), and H19 ICR (D) in individual oocytes derived from low dosage (L, 6.25 IU) superovulated B6(CAST7p6)XB6 females. For each gene, n = 15. Details are as described in Figure 1. Fertility and Sterility 2011 96, 734-738.e2DOI: (10.1016/j.fertnstert.2011.06.055) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Methylation analysis of the Snrpn ICR (A), Kcnq1ot1 ICR (B), Peg3 DMR (C), and H19 ICR (D) in individual oocytes derived from high dosage (H, 10 IU) superovulated B6(CAST7p6)XB6 females. For each gene, n = 15–17. Details are as described in Figure 1. Fertility and Sterility 2011 96, 734-738.e2DOI: (10.1016/j.fertnstert.2011.06.055) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Methylation analysis of individual oocytes without cumulus cell contamination. Only a single strand of oocyte DNA was expected to amplify, either a methylated B6 or CAST Snrpn, Kcnq1ot1, or Peg3 allele and either an unmethylated B6 or CAST H19 allele. For each sample, five clones were sequenced. Oocytes (indicated to left) with a single methylation pattern, a single genotype (B6 or CAST), and identical non-CpG conversion pattern (percentage indicated to the right) were included in the analysis. Representative oocytes shown. Black circles indicate methylated CpGs. White circles indicate unmethylated CpGs. Fertility and Sterility 2011 96, 734-738.e2DOI: (10.1016/j.fertnstert.2011.06.055) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Methylation analysis of individual oocytes with cumulus cell (CC) contamination and/or polar body (PB) inclusion. Expected methylation patterns for females and their oocytes for Snrpn, Kcnq1ot1, and Peg3 (top left) and H19 (top right) are shown. Only a single strand of oocyte DNA was expected to amplify, either a methylated CAST (CM) or B6 (BM) Snrpn, Kcnq1ot1, or Peg3 allele and either an unmethylated CAST (CU) or B6 (BU) H19 allele. For each sample, five clones were sequenced. Oocytes (designation indicated to left) with multiple methylation patterns, both genotypes (B6, B, and CAST, C), and/or multiple non-CpG conversion patterns (percentage indicated to the right) indicative of multiple strand amplification, were excluded from the analysis. Representative oocytes shown. Black circles indicate methylated CpGs. White circles indicate unmethylated CpGs. Fertility and Sterility 2011 96, 734-738.e2DOI: (10.1016/j.fertnstert.2011.06.055) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions