The oocyte spindle is preserved by 1,2-propanediol during slow freezing  Ching-Chien Chang, Ph.D., Li-Ying Sung, Ph.D., Chih-Jen Lin, M.Sc., Hilton I. Kort, M.D., Xiangzhong Yang, Ph.D., X. Cindy Tian, Ph.D., Zsolt Peter Nagy, M.D., Ph.D.  Fertility and Sterility  Volume 93, Issue 5, Pages 1430-1439 (March 2010) DOI: 10.1016/j.fertnstert.2009.01.106 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Influence of the freezing process on the MII spindle. Confocal images of α-tubulin (A,D), chromatin (B,E), and merge of chromatin and α-tubulin (C,F) of representative oocytes treated with freezing solution for 20 minutes at RT (n = 35; A–C) and oocytes that have been fixed immediately upon thawing (without removal of cryoprotectants) (n = 31; D–F). Oocytes have been frozen and thawed display stabilized MII spindle with chromosomes aligned on the metaphase plate (D–F) resembling oocytes before the freezing process (A–C). Fertility and Sterility 2010 93, 1430-1439DOI: (10.1016/j.fertnstert.2009.01.106) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The effect of cryoprotectants on preserving the MII spindle during low-temperature treatment (4°C for 30 minutes). Confocal images of α-tubulin (A,D,G,J), chromatin (B,E,H,K), and merge of chromatin and α-tubulin (C,F,I,L) of representative oocytes treated with choline-based freezing solution without PROH and sucrose (A–C), choline-based freezing solution with 0.3 M sucrose but without PROH (D–F), choline-based freezing solution with 1.5 M PROH but without sucrose (G–I), and complete freezing solution with both 1.5 M PROH and 0.3 M sucrose (J–L). In the absence of PROH and sucrose, oocytes display disorganized MII spindle after low-temperature treatment (A–C). The 1.5 M PROH stabilized MII spindle during low-temperature treatment (G–I and J–L). However, sucrose alone was not able to prevent the spindle disassembly caused by low-temperature treatment, and those oocytes revealed diminished microtubule apparatus with two distinctive spindle poles (D–F). Fertility and Sterility 2010 93, 1430-1439DOI: (10.1016/j.fertnstert.2009.01.106) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 MII spindle organization during the thawing processes. Confocal images of α-tubulin (A,D,G,J), chromatin (B,E,H,K), and merge of chromatin and α-tubulin (C,F,I,L) of cryopreserved oocytes first thawing in 0.5 M sucrose solution at RT for 10 minutes (A–C), secondly transferred into 0.2 M sucrose solution at RT for another 10 minutes (D–F), and finally washed by the HEPES-buffered solution for 10 minutes at RT (G–I). The cryopreserved oocytes were directly thawed into freezing solution with 1.5 M PROH and 0.3 M sucrose for 30 minutes at RT (without removal of any cryoprotectants) (J–L). The MII spindle started to disassemble following thawing, and it is noteworthy that the microtubule apparatus was detached from the metaphase plate when the cryoprotectants have been completely removed (I). On the other hand, the disorganized MII spindle was not avoidable when the cryopreserved oocytes were directly thawed into freezing solution with complete cryoprotectants (J–L). Fertility and Sterility 2010 93, 1430-1439DOI: (10.1016/j.fertnstert.2009.01.106) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 MII spindle underwent disassembling and reassembling during the first hour after thawing. Confocal images of α-tubulin (A,D), chromatin (B,E), and merge of chromatin and α-tubulin (C,F) of cryopreserved oocytes fixed after the complete thawing processes (after removal of cryoprotectants) and subsequent incubation for 30 minutes at 37°C. Approximately, one-third of the thawed oocytes (9/29) did not have detectable microtubule structure surrounding chromosomes (A–C), and the rest of oocytes (20/29) revealed that metaphase chromosomes were attached with scarce microtubule structure (D–F). Fertility and Sterility 2010 93, 1430-1439DOI: (10.1016/j.fertnstert.2009.01.106) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Nuclear and microtubule dynamics of frozen and thawed oocytes after incubation at 37°C. Confocal images depict microtubules (green) and chromatin (red). (A) A representative oocyte reveals a well-organized bipolar spindle. (B) A representative oocyte lacks detectable microtubule structure. (C) A representative oocyte exhibits telophase of meiotic spindle with two sets of separated chromosomes. (D) A representative oocyte reveals an early stage pronucleus in the cytoplasm. Arrow indicates the pronucleus. Fertility and Sterility 2010 93, 1430-1439DOI: (10.1016/j.fertnstert.2009.01.106) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions