PHASE-TRANSITION TEMPERATURE AND CHILLING SENSITIVITY OF BOVINE OOCYTES

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock , is defined as an irreversible damage expressed shortly after exposur e to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between D CI in sperm and the thermotropic phase transition of their membrane li pids was demonstrated In the present study, we examined the phase tran sition of the membrane lipids of immature and in vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20 degrees C, whil e a very broad phase transition, which centered around 10 degrees C, w as observed for mature oocytes (MII) stage. Thermotropic phase transit ions were demonstrated to be related to the temperature at which DCI a ffected the integrity of the oocyte membranes. When immature oocytes w ere cooled to 13 degrees C, fewer oocytes (40%) retained their membran e integrity than after exposure to 4 degrees C (51%) or holding them a t 38 degrees C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differenc es in membrane integrity were observed when in vitro-matured oocytes w ere cooled to the same temperatures. Subsequently, GV oocytes were coo led to 4 degrees C, and 26% underwent maturation and 19% underwent fer tilization in vitro. In vitro-matured oocytes that were cooled to 4 de grees C displayed a slightly decreased rate of fertilization; the over all fertilization was 60% with 24% polyspermy, rather than the 76% fer tilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes . Nucleated bovine GV oocytes were electrofused with in vitro-matured and enucleated oocytes, and then cooled to 4 degrees C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibilit y to low temperatures. This finding led to an improvement in the survi val of oocytes after cryopreservation. (C) 1996 Academic Press, Inc.