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 containing 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 assessed 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 rupture or spindle disorganization between 200 and 1800 milliosmolal. Higher levels of osmotic stress caused rupture 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.

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

“…Thus, knowing the osmotic tolerance limits is essential for optimizing cryopreservation methods. Previously, Mullen et al 46 demonstrated that MII spindle damage in bovine oocyte occurs more frequently as the extracellular solution concentrations diverge from isosmotic. Moreover, they estimated that to prevent osmotic damage to the MII spindle with a probability of 90%, it is necessary to use CPA addition and removal procedure which maintains the cells within a volume range of 1.1 to 0.52 times the isotonic volume.…”

“…However, during CPA removal, the non-dilute model predicts that GV and MII oocytes will shrink to an equilibrium value much faster than the 2P model predicts, which involves maintaining the cells in a more prolonged state of osmotic stress. Based on the osmotic damage model developed by Mullen et al 46 , maintenance of the oocytes at a volume of 38% relative to isotonic is expected to cause about 35% of oocytes to experience osmotic damage to the spindle.…”

Investigation of methods and factors influencing mouse oocyte quality, in vitro fertilization and embryo development

“…Although it has been widely used, the vitrification procedure can have many adverse effects on oocytes including altered F-actin distribution (108), premature cortical granules exocytosis (PCGE) (172), change in the mitochondria membrane potential (22,175,181), disruption of the mitochondria distribution (176) and subsequent cell death (182). The origins of these adverse effects have been mainly attributed to osmotic shock due to sudden exposure of oocytes to high concentrations of vitrification solution (183,184) and sudden cold shock (185), chemical toxicity (183) and the relative large size of oocyte (70-120 μm) and thus, high water content in the oocyte (186)(187)(188).…”