Effects of vitrification solutions and equilibration times on the morphology of cynomolgus ovarian tissues  Shu Hashimoto, Nao Suzuki, Masaya Yamanaka, Yoshihiko Hosoi, Bunpei Ishizuka, Yoshiharu Morimoto  Reproductive BioMedicine Online  Volume 21, Issue 4, Pages 501-509 (October 2010) DOI: 10.1016/j.rbmo.2010.04.029 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Vitrification of ovarian cortex. (A) Section of ovarian cortex of parous cynomolgus monkey. (B) Cryosupport. (C) Vitrified ovarian cortex with Cryosupport. Reproductive BioMedicine Online 2010 21, 501-509DOI: (10.1016/j.rbmo.2010.04.029) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Light microscopic images of ovarian tissues, non-frozen (control), vitrified after 5min exposure to 5.64mol/l ethylene glycol+5% (w/v) polyvinylpyrrolidone+0.5mol/l sucrose (VSEGP5) and vitrified after 5min exposure to 3.22mol/l ethylene glycol+2.56mol/l dimethylsulphoxide+0.5mol/l sucrose (VSED5). In VSEGP5, most of oocytes were surrounded by compact ovarian stroma similarly to control and a few oocytes were slightly damaged. In VSED5, there a lot of oocytes damaged severely. Black arrow=severe damage; white arrow=slight damage. Original magnification ×20. Bar=100μm. Reproductive BioMedicine Online 2010 21, 501-509DOI: (10.1016/j.rbmo.2010.04.029) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Effect of vitrification solutions on ovarian tissues. (A) Non-frozen primordial–primary follicle. (B) Primordial follicle vitrified after 5min exposure to 5.64mol/l ethylene glycol+5% (w/v) polyvinylpyrrolidone+0.5mol/l sucrose (VSEGP). (C) Primary follicle vitrified after 20min exposure with 3.22mol/l ethylene glycol+2.56mol/l dimethylsulphoxide+0.5mol/l sucrose (VSED). (A1) Oocyte surrounded by a single layer of flattened or cuboidal follicle cells. (B1) Oocyte surrounded by a single layer of flattened follicle cells. (C1) Oocyte surrounded by a single layer of cuboidal follicle cells. The cytoplasmic organelles are clustered in the oocyte cytoplasms. Both non-vitrified (A2) and vitrified cortex (B2 and C2) consist of a dense matrix of collagen bundles (white arrow; A2, B2 and C2) and granulosa cells (GC) containing peripheral heterochromatin in their nuclei and numerous rod-shaped mitochondria scattered in their cytoplasm. Change in shape of nuclear membrane was observed and a few follicle cells were damaged (arrow) after vitrification (C1). A continuous basement membrane is present at the periphery of the follicle (black arrow; A2, B2 and C2). Healthy-looking follicles show the oocyte cytoplasm with an abundance of mitochondria (mt) or the germinal vesicle (GV). Granulosa-cells show distinct cell membranes (m) and nuclei (n). (C2) Vitrified oocyte showing vacuolated mitochondria (Vmt), lipofuchin body (lb) and lysosomes (lys). Original magnification: A1=×2500; B1=×3000; C1=×2000; A2, B2, C2=×15,000. Bars: A1, B1, C1=10μm; A2, B2, C2=2μm. Reproductive BioMedicine Online 2010 21, 501-509DOI: (10.1016/j.rbmo.2010.04.029) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Effect of vitrification solutions on follicles. (A1–3) Primordial–primary follicle and (B1–3) primordial follicle, vitrified after 10 or 20min exposure with 3.22mol/l ethylene glycol+2.56mol/l dimethylsulphoxide+0.5mol/l sucrose (VSED). (A1) Oocyte surrounded by a single layer of flattened or cuboidal follicle cells. (B1) Severely damaged oocyte surrounded by a layer of flattened follicle cells. The space between follicle cells and stromal cells was spread, presumably because of vitrification procedure in both cases. The cytoplasmic organelles are clustered in the oocyte cytoplasm. The damage of organelles in oocyte cytoplasm was low level (A1). Vitrified cortex consisting of a dense matrix of collagen bundles (arrow), granulosa cells containing peripheral heterochromatin in their nuclei (n) and rod-shaped mitochondria (mt) scattered in their cytoplasm (A2). A continuous basement membrane is also present at the periphery of the follicle (white arrow). The mitochondria (mt) with a low-density matrix, vacuolated mitochondria (Vmt) and a Golgi apparatus (g) (A3). (B1–3) Severely damaged oocyte cytoplasm contains a lot of vacuoles and vacuolated mitochondria. A continuous basement membrane is present at the periphery of the follicle (white arrow). (B2) Cytoplasmic organella are vacuolated in the oocyte cytoplasm after vitrification and warming. (B3) Vacuolated mitochondria and lysosomes (lys) in vitrified oocyte. (C) Autolysosome formation to eliminate damaged organella in oocytes vitrified after 10min exposure with 5.64mol/l ethylene glycol + 5% (w/v) polyvinylpyrrolidone + 0.5mol/l sucrose. Original magnification: A1=×1500; B1=×2500; A2=×15,000; B2=×5000; A3, B3=×25,000; C=×50,000. Bars: A1=20μm; B1=10μm; A2=2μm; B2=5μm; A3, B3=1μm; C=0.5μm. Reproductive BioMedicine Online 2010 21, 501-509DOI: (10.1016/j.rbmo.2010.04.029) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions