Modeling the Probability of MII Spindle Disruption in Bovine Oocytes as a Function of Total Osmolality Using Logistic Regression and Its Application toward Improved CPA Addition and Removal Procedures

Abstract

During cryopreservation, cells undergo volume excursions as a result of exposure to anisosmotic solutions con- taining cryoprotective agents. Cryopreservation methods can be engineered to maintain cell volumes within their osmotic tolerance limits. In this study we investigated the osmotic tolerance limits of the bovine oocyte oolemma and metaphase II spindle. Oocytes were exposed to solutions (75- 3000 milliosmolal) for 10 min, then cultured for 1 h. Oolemma integrity was assessed visually prior to fixation. The structure of the metaphase II spindle was as- sessed using immunocytochemistry and laser scanning confocal microscopy. Logistic regression was used to model the probability of spindle disruption as a function of solution osmolality. Few cells experienced oolemma rup- ture or spindle disorganization between 200 and 1800 milliosmolal. Higher levels of osmotic stress caused rup- ture of the oolemma and disruption of the spindle structure. These results can be applied to improve methods for the addition and removal of cryoprotective agents that will protect the spindle from osmotic damage.