Fundamental cryobiology of mammalian oocytes

The cryopreservation of mammalian oocytes from various species is becoming an integral part of assisted reproductive technologies in the area of animal reproduction, human reproductive medicine, research, and conservation of rare genetic stocks. Maintenance of the integrity of the oocyte during cryopreservaton procedure is crucial for the normal fertilization and further development following thawing. The limited cryosurvival of mammalian oocytes is mainly due to insufficient information on the biophysical, physiological and ultrastructural properties of the oocytes, which are cryobiologically important. Fundamental changes take place during oocyte development from germinal vesicle (GV) to metaphase II (MII) stage at both cytoplasmic and plasma membrane level. These changes may also affect plasma membrane permeability characteristics that are cryobiologically important. The hypothesis is that the osmotic behavior of oocytes to water and cryoprotective agents (CPAs) may be stage and species specific and thus may require species and stage specific protocols. In order to test the hypothesis, osmotic behavior of GV and MII stage mouse, rat and bovine oocytes was studied in the presence of CPA. These were the determination of: (1) oolemma temperature-dependent hydraulic conductivity (L$\rm\sb{p}),$ cryoprotectant permeability (P$\rm\sb{CPA})$ and their reflection coefficients $(\sigma)$ as well as activation energies for such permeability parameters, and (2) osmotic tolerance limits. These values were then incorporated in the calculation of: (1) optimal procedures for the addition and removal of CPA and its intracellular concentration, (2) cooling rates necessary to minimize intracellular ice formation as well as so-called solution effects by estimating water loss kinetics of oocytes during freezing. The data obtained from this dissertation support the initial hypothesis that osmotic behavior of oocytes from different mammalian species and developmental stages are fundamentally different. Cryopreservation protocol designed for a specific species and developmental stage may not be as successful when it is applied to the others. Therefore, species and developmental stage specific protocols may be necessary to improve cryosurvival of oocytes. In conclusion, it can be suggested that there is a great deal to be learned from a comparative study of the fundamental cryobiology of oocytes from various mammalian species, and understanding the characteristics that are unique to each