Determination of oocyte membrane permeability coefficients and their application to cryopreservation in a rabbit model.
Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (V(b)), hydraulic conductivity (L(p)), permeability (P(s)) to dimethylsulfoxide (Me(2)SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1x PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of L(p) and P(s). Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van't Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7+/-1.2 microm (n=16). The rabbit oocyte exhibited an "ideal" osmotic response in the range from iso-osmolity to 1200 mOsm. The V(b) was determined to be 20% of the isotonic value with r(2)=0.97. The values of L(p) were determined to be 0.79+/-0.26, 0.82+/-0.22, and 0.64+/-0.16 microm min(-1)atm(-1) and the P(s) values were determined to be 2.9+/-1.3, 2.7+/-1.3, and 0.27+/-0.18x10(-3) cm min(-1) for Me(2)SO, EG and GLY, respectively. There were no significant differences (p>0.05) between values for L(p) and P(S) in the presence of the Me(2)SO and EG. However, these values were significantly different from the values in presence of GLY. We calculated the AOD values of those oocytes that experienced the process of CPA additions/dilutions and found that these values were highly correlated to the development rates of these oocytes after parthenogenetic activation (r=-0.98).
Liu
Jun
J
Agricultural Biotechnology Center, Gödöllo, Hungary.
Mullen
Steve
S
Meng
Qinggang
Q
Critser
John
J
Dinnyes
Andras
A
D000818:Animals; D002463:Cell Membrane Permeability; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000378:metabolism / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000378:metabolism / Q000494:pharmacology; D019855:Ethylene Glycol / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D005990:Glycerol / Q000378:metabolism / Q000494:pharmacology; D023421:Models, Animal; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D009994:Osmolar Concentration; D011817:Rabbits
D003451:Cryoprotective Agents; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide