Effect of postovulatory oocyte aging on DNA methylation imprinting acquisition in offspring oocytes  Xing-Wei Liang, Ph.D., Zhao-Jia Ge, M.S., Lei Guo, Ph.D., Shi-Ming Luo, Ph.D., Zhi-Ming Han, Ph.D., Heide Schatten, Ph.D., Qing-Yuan Sun, Ph.D.  Fertility and Sterility  Volume 96, Issue 6, Pages 1479-1484 (December 2011) DOI: 10.1016/j.fertnstert.2011.09.022 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 DNA methylation status of DMRs in oocytes from aged-oocyte offspring analyzed by COBRA. Oocytes from aged-oocyte F1 offspring were recovered. Samples of five oocytes were subjected to each individual bisulfite-treatment and PCRs. Imprinted genes (A) Peg3, (B) Snrpn, (C) Peg1, and (D) H19 were amplified through nested PCR using bisulfite-treated DNA as a template. The second-round specific amplification products were digested with appropriate endogenous restriction enzymes as indicated on the left. PCR products of bisulfite-treated liver DNA were also digested with appropriate enzymes and used as a control (around 50% methylation vs. 50% unmethylation for cut and uncut bands, respectively). Uncut and cut denote unmethylated and methylated CpG loci, respectively. The numbers on the figure’s top indicate the number of individual bisulfite treatments and PCRs. The same symbols apply to Figures 2 and 3. Fertility and Sterility 2011 96, 1479-1484DOI: (10.1016/j.fertnstert.2011.09.022) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 DNA methylation status of DMRs in oocytes from aged-oocyte offspring analyzed by BS. The same bisulfite-treated DNA amplified by PCR and used for Figure 1 was assayed by sequencing. According to the COBRA results shown in Figure 1, (A) for Peg3, nos. 13 and 19, which had bands similar to the bisulfite-treated liver DNA control digested by the same enzymes, were pooled; all the remaining individual PCR products displaying different bands compared with those of bisulfite-treated liver DNA control were pooled; (B) for Snrpn, nos. 1, 4, 8, and 17 with a band similar to that of bisulfite-treated liver DNA digested by BstUI were pooled, and all the remaining individual PCR products with different bands compared with bisulfite-treated liver DNA were pooled, respectively; (C) for Peg1, all individual PCR products only had cut fragments, and all of them were pooled; (D) for H19, all individual PCR products were uncut by both TaqαI and RsaI, and all of them were pooled. The pooled products were subcloned and sequenced. Individual lines, clones sequenced; filled circles, methylated cytosines; open circles, unmethylated cytosines. The CpG loci that were also assayed by the restriction enzymes used in the COBRA are indicated at the top. The same symbols apply to Figure 3. Fertility and Sterility 2011 96, 1479-1484DOI: (10.1016/j.fertnstert.2011.09.022) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 DNA methylation status of Peg3 DMR in oocytes from fresh-oocyte offspring analyzed by (A) COBRA and (B) BS. COBRA results showed that all products were digested by both TaqαI and BstUI, so all of them were pooled and subjected to sequencing. Fertility and Sterility 2011 96, 1479-1484DOI: (10.1016/j.fertnstert.2011.09.022) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions