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Abstract
Vitrification of mammalian oocytes employs hypertonic solutions of cryoprotectant additives (CPAs), causing a rapid loss of water from the cell. This severe dehydration can result in osmotic shock. Oocytes are known to be altered by abrupt changes in osmotic pressure (1987 Cryobiology 24, 387–402), which may result in ultrastructural changes. Therefore, the objective of this study was to examine the effects of severe dehydration of mature bovine oocytes on the cytoskeletal arrangement of microtubules and actin filaments as well as the metaphase spindle prior to vitrification. In Experiment I, oocytes (n=30) obtained from Ovagenix (San Angelo, TX, USA) were sequentially exposed (5 or 6/treatment group) to increasing hypertonic solutions of two saccharides (0.15, 0.3, 0.5, 0.65, 0.99M sucrose and 0.125, 1.25, 0.35, 0.55, 0.65M trehalose) prepared in TCM-199 or M2 medium. Control oocytes (n=22) were subjected to isotonic control solutions (TCM-199 and M2 alone). Relative average volumes of oocytes were determined as previously described and then Boyle vant Hoff plots were constructed. In this study, bovine oocytes behaved similarly when exposed to increasing osmolalities of sucrose and trehalose in both media. The oocytes exhibited a linear decrease in relative volume as a function of 1/(osmotic pressure)to a minimum of approximately 32% of the isotonic volume for 0.99M sucrose in M2 medium (r2=0.9785) (Graphpad 2.04, GraphPad Software, San Diego, CA, USA). In the second experiment, partially denuded bovine oocytes (n=106) were sequentially dehydrated to determine the effect of dehydration on cytoskeletal structures and the meiotic spindle. Oocytes were suspended in solutions of 0.99M sucrose or 0.65M trehalose in either TCM-199 or M2 medium (concentration of sucrose and trehalose where maximum dehydration occurred in the first experiment). Correspondingly, similar oocytes (n=30) were allotted to either TCM-199 or M2 medium control groups. Oocytes were subsequently fixed, immunolabelled for tubulin (rat anti-α-yeast tubulin and anti-rat FITC) and actin (rhodamine phalloidin), stained for DNA (Hoechst 33342), and then mounted for analysis. Cytoskeletal arrangement and DNA localization were visualized using fluorescence and laser-scanning microscopy. Microtubules in control oocytes were intimately associated with the meiotic spindle, which appeared as a symmetrical, barrel-shaped structure with anastral poles. Intact spindles were observed in 63% of the control oocytes compared with 0% in all treatment groups, with the exception of 3% of the oocytes suspended in 0.99M sucrose in TCM-199. Co-localization of actin with tubulin was observed in all oocytes with intact spindles. In conclusion, bovine oocytes respond as perfect osmometers when suspended in hypertonic solutions of saccharides. Oocytes exposed to such solutions exhibited extensive disruption of their meiotic spindles, which would undoubtedly result in reduced fertilization and abnormal development. Further study is needed to determine whether the effects of dehydration can be reversed by sequential rehydration.
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