Weihong Hu, M. D. , Ph. D. , Dennis Marchesi, Ph. D. , Jie Qiao, M. D

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Effect of slow freeze versus vitrification on the oocyte: an animal model Weihong Hu, M.D., Ph.D., Dennis Marchesi, Ph.D., Jie Qiao, M.D., Ph.D., Huai L. Feng, Ph.D., H.C.L.D., E.L.D. Fertility and Sterility Volume 98, Issue 3, Pages 752-760.e3 (September 2012) DOI: 10.1016/j.fertnstert.2012.05.037 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effects of different cryopreservation protocols on spindle configuration in the bovine oocyte. The normal configurations of spindle and chromosomal alignment in bovine MII oocytes (A) and abnormal spindles with disorganized microtubules or multipoles, along with scattered chromosomes, were considered abnormal (B). *P<.05. Fertility and Sterility 2012 98, 752-760.e3DOI: (10.1016/j.fertnstert.2012.05.037) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of different cryopreservation protocols on global DNA methylation status in bovine oocytes. Nucleus DNA (A); DNA methylation (B); nucleus DNA and DNA methylation merge (C). *P<.05. Fertility and Sterility 2012 98, 752-760.e3DOI: (10.1016/j.fertnstert.2012.05.037) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effects of different cryopreservation protocols on apoptosis in bovine oocytes and COCs. COC without any cumulus cell apoptosis (A); COC with <15% cumulus cells apoptosis (B); COC with 15%–70% cumulus cells apoptosis (C); COC with >70% cumulus cells apoptosis (D). *P<.05. Fertility and Sterility 2012 98, 752-760.e3DOI: (10.1016/j.fertnstert.2012.05.037) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Apoptosis-related DNA fragmentation detected by TUNEL assay in bovine oocytes. Nucleus DNA (A); DNA fragment with apoptosis (B); merge image of nucleus DNA and DNA fragment with apoptosis (C). *P<.05. Fertility and Sterility 2012 98, 752-760.e3DOI: (10.1016/j.fertnstert.2012.05.037) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Effects of different cryopreservation protocols on cortical granules (CGs) distribution. Distributional patterns of the CGs were classified into three types in mature bovine oocytes: type I, CGs distributed in clusters (A); type II, CGs dispersed and partly clustered (B); and type III, all CGs dispersed (C). P>.05. Fertility and Sterility 2012 98, 752-760.e3DOI: (10.1016/j.fertnstert.2012.05.037) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Effects of different cryopreservation protocols on mitochondria distribution. Representative micrographs of mitochondrial localization in all categories of mature oocytes were classified into three types: category 1 oocytes displayed only small numbers of mitochondrial clumps in a peripheral location (A); category 2 (B) was between category 1 and category 3; category 3 oocytes showed the strongest and most homogeneous staining (C). *P<.05. Fertility and Sterility 2012 98, 752-760.e3DOI: (10.1016/j.fertnstert.2012.05.037) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions