Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (Lp), cryoprotectant permeability (P[CPA]), and reflection coefficient (sigma) for immature (germinal vesicle stage, GV) and in vitro-matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 +/- 2 degrees C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The Lp values for GV and MII oocytes in DMSO (L(p)DMSO) were 0.70 +/- 0.06 and 1.14 +/- 0.07 microm/min/atm (mean +/- SEM) and in EG (L(p)EG) were 0.50 +/- 0.06 and 0.83 +/- 0.07 microm/min/atm, respectively. Estimates of P(DMSO) for GV and MII oocytes were 0.36 +/- 0.03 and 0.48 +/- 0.03 microm/sec, and PEG values for GV and MII oocytes were 0.22 +/- 0.03, 0.37 +/- 0.03 microm/sec, respectively. The values for GV and MII oocytes in DMSO (sigma[DMSO]) were 0.86 +/- 0.03 and 0.90 +/- 0.04 and in EG (sigma[EG]) were 0.94 +/- 0.03 and 0.76 +/- 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte P(DMSO) is higher than the P(EG). These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages.