Human oocyte cryopreservation and the fate of cortical granules Yehudith Ghetler, Ph.D., Ehud Skutelsky, Ph.D., Isaac Ben Nun, M.D., Liah Ben Dor, M.Sc., Dina Amihai, M.Sc., Ruth Shalgi, Ph.D.  Fertility and Sterility  Volume 86, Issue 1, Pages 210-216 (July 2006) DOI: 10.1016/j.fertnstert.2005.12.061 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Detection of cortical granule exocytosis (CGE) by labeling with biotinylated lectin Lens culinaris agglutinin and Texas red–strepavidin. Composite confocal microscopy images (A–D) and light microscopy images (a–d) of untreated mature oocyte (A, a), untreated 3PN zygote (B, b), frozen-thawed (slow freezing–rapid thawing in 1,2 propanediol) mature oocyte (C, c), and frozen-thawed (slow freezing–rapid thawing in 1,2 propanediol) germinal vesicle stage oocyte (D, d), illustrating the degree of CGE at the various treatments. Bar = 35 μm. Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 86, 210-216DOI: (10.1016/j.fertnstert.2005.12.061) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 The effect of cryopreservation on mature oocytes. (Top) An untreated oocyte: Abundant dark electron-dense granules (white arrow) are present in the cortex, and light (black arrow) granules are present mainly deeper in the cytoplasm. (Bottom) A frozen-thawed oocyte: Very few granules are present at the cortex. Light (black arrow) and a few dark (white arrow) granules are in the inner region. A large number of membrane-coated transparent vesicles (V) are observed, scattered throughout the ooplasm. ZP = zona pellucida. Bar = 2 μm. Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 86, 210-216DOI: (10.1016/j.fertnstert.2005.12.061) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 The effect of cryopreservation on immature (geminal vesicle stage) oocytes. (Top) An untreated oocyte: Dark electron-dense granules (white arrow) are present in the cortex. Numerous aggregated light (black arrow) granules appear throughout the cytoplasm. (Bottom) A frozen-thawed oocyte: Very few dark (white arrow) granules are present at the cortex. Light (black arrow) aggregated granules are in the inner cytoplasm. A large number of membrane-coated transparent vesicles (V) were observed scattered throughout the ooplasm. ZP = zona pellucida. Bar = 2 μm. Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 86, 210-216DOI: (10.1016/j.fertnstert.2005.12.061) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions