Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment  Claudia Messias Gomes, M.D., Cristine Ane Silva E. Silva, M.S., Nicole Acevedo, Ph.D., Edmund Baracat, M.D., Ph.D., Paulo Serafini, M.D., Gary D. Smith, Ph.D.  Fertility and Sterility  Volume 90, Issue 4, Pages 1396-1404 (October 2008) DOI: 10.1016/j.fertnstert.2007.08.025 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The closed pulled straw (CPS) method for oocyte vitrification. (A) The tip of the pulled straw has four marked lines. To load the pulled straw, VS is aspirated to the first line, and then oocytes and VS are aspirated to reach the second line; additional VS is aspirated so that the fluid meniscus reaches the third line. The relevant characteristics of CPS are its small, fine, and delicate nature. To show this, CPS is compared with the size of a penny in the figure. The straw is attached to a 1-mL syringe. The metal end is where the oocytes will be placed. (B) A CPS containing seven fresh oocytes. (C) CPS is heat sealed just below the first mark and then above the fourth mark. At warming, CPS is taken out of the liquid nitrogen and is cut at the large end near the fourth mark. Fertility and Sterility 2008 90, 1396-1404DOI: (10.1016/j.fertnstert.2007.08.025) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Recovery and survival rates of vitrified oocytes after warming. The rates are not significantly different between straws with two or 10 oocytes. Recovery after warming: 100% (two oocytes per straw) and 95% ± 4% (10 oocytes per straw). Survival after warming: 95% ± 3% (two oocytes per straw) and 98% ± 3% (10 oocytes per straw). (B) Assessment of oocyte survival using light-microscope analysis with Hoffman Modulation Contrast optics (Leica DMIRB; Leica Microsystems) at ×400 magnification. Fertility and Sterility 2008 90, 1396-1404DOI: (10.1016/j.fertnstert.2007.08.025) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Observations on influence of vitrification/warming solution alone (Sol Expos), cryopreservation with vitrification/warming (Vitr), and time of assessment after warming on spindle dynamics. Depolymerization of the spindles occurred in vitrified and warmed oocytes fixed immediately after removal from the final WS (T0) but not in oocytes exposed to vitrification/warming solutions alone. After 37°C culture for 2 hours (T2), β-tubulin repolymerized and oocytes vitrified/warmed had normal-appearing MII spindles, similar to oocytes exposed to vitrification/warming solutions alone. Micrographs were obtained with a Leica DMR fluorescent microscope at ×400 magnification. PB = polar body. Fertility and Sterility 2008 90, 1396-1404DOI: (10.1016/j.fertnstert.2007.08.025) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Representative time-lapse micrographs demonstrating the temporal configuration of MII oocyte spindles after vitrification and warming. Oocytes were vitrified and warmed as described in Material and Methods. T0 represents initial observations after removal of oocytes from the final WS. These real-time observations are supportive of data gathered in fixed oocytes at T0 and T2. Original magnification, ×400. Fertility and Sterility 2008 90, 1396-1404DOI: (10.1016/j.fertnstert.2007.08.025) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 5 β-tubulin immunocytochemical analysis in oocytes isolated as controls (control), after solution exposure only (Sol Expos) or after vitrification/warming (Vitr) and 2-hour culture at 37°C. (A) Example of strict criteria used for spindle morphology assessment. (B) Spindle length was not significantly different between treatment groups. Leica DMR fluorescent microcope at ×400 magnification. Fertility and Sterility 2008 90, 1396-1404DOI: (10.1016/j.fertnstert.2007.08.025) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions