1. Effects of cryoprotectants and cryopreservation on germinal vesicle-stage cumulus– oocyte complexes of rhesus monkeys 
Catherine A. VandeVoort, Ph.D., Cynthia R. Shirley, Ph.D., Dana L. Hill, B.S., S.P. Leibo, Ph.D. 
Fertility and Sterility 
Volume 90, Issue 3, Pages (September 2008)
DOI: /j.fertnstert
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Control and cryopreserved rhesus COCs. The complexes were stained with Alexa 488-phalloidin that binds to f-actin, and with monoclonal antibodies to α- and β-tubulin followed by Cy-3 antimouse IgG. The images on the left are of the f-actin staining (green), and the images on the right are of tubulin (red). To expand the range of brightness values so as to increase contrast, the digital images were subjected to a histogram stretch on the red channel. A magnification scale bar of 20 μm is shown on each image. (A) Control COC that was fixed immediately after collection. The arrow points to the germinal vesicle in the left-hand image and to the nucleolus shadow in the right-hand image. There were cumulus cells completely surrounding the oocyte. (B) Handled COC that was exposed to 1.5 mol/L PG mol/L sucrose but was not cooled or frozen. There is not an obvious germinal vesicle in this oocyte, although there is a shadow of the nucleolus (arrow). (C) Cumulus–oocyte complex that was subjected to slow freezing in a solution of 1.5 mol/L PG + sucrose and then was fixed immediately after being thawed. This image illustrates a COC that was relatively undamaged by the treatment in that it retained its spherical shape, although the microtubules were missing and there was a noticeable decrease in the amount of actin in the cytoplasm. A shadow of the nucleolus is indicated by the arrow. (D) Another COC subjected to slow freezing that was fixed immediately after being thawed. This image illustrates a badly damaged COC; most of the cumulus cells have been stripped off, and the intracellular structure has been lost.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Control and cryopreserved rhesus COCs. As in Figure 1, the complexes were stained to reveal f-actin (left) and to reveal tubulin (right). A magnification scale bar of 20 μm is shown on each image. (A) Control COC that was fixed immediately after being collected. This picture was taken toward the surface of the oocyte so that the internal actin pattern is less evident. There were cumulus cells completely surrounding the oocyte. (B) Handled COC that was exposed to 2.2 mol/L DMSO mol/L EG mol/L sucrose but was not cooled or cryopreserved. A very prominent germinal vesicle was evident in the oocyte (a). Transzonal processes were also present, as was tubulin, although some of these appeared to be broken (arrowheads). (C) Rhesus COC that was cryopreserved by vitrification. After being warmed and liquefied, the COC was diluted out of the cryoprotectant and immediately fixed. Transzonal processes were evident, although many of the cumulus cells had been stripped off the zona. The fine-structured tubulin was not evident. (D) Another COC that was cryopreserved by vitrification. After being warmed and liquefied, the COC was cultured for 3 hours and then was fixed. This image was taken at the same magnification as those above, indicating that the entire COC had contracted in culture. Almost all of the cumulus cells had been stripped off, and all transzonal processes were missing.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Control and cryopreserved rhesus COCs. As in Figures 1 and 2, these complexes were fixed and stained to reveal tubulin. A magnification scale bar of 10 μm is shown on each image. (A) Control COC that shows the fine tubulin “netting” within the cytoplasm. Arrowhead points to a transzonal process. (B) Rhesus COC that was cryopreserved by vitrification. The arrows define the edges of the zona pellucida. The intracellular tubulin is missing. (C) Rhesus COC cryopreserved by slow cooling. As in (B), the arrows define the edges of the zona pellucida, and the intracellular tubulin is missing.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions