DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a novel preservation technique for primate oocytes. Oocytes are unique totipotent cells that offer exceptional prospects for studying fundamental developmental biology, as successfully explored in many areas including nuclear reprogramming and derivation of embryonic stem cells. Therefore, successful banking of oocytes from different model species would enormously contribute to the advancement of science, medical technologies, and conservation of endangered species. A cryopreservation method developed and thoroughly tested using non-human primate oocytes would not only facilitate basic research and conservation of endangered primates but would also be a major stepping-stone towards a reliable preservation protocol for human oocytes for infertility treatment. To this end, we propose a mechanistic novel approach based on survival schemes in nature where a variety of organisms such as frogs, tardigrades, and brine shrimp can survive freezing or almost complete drying. Accumulation of sugars is extremely critical for their survival strategy. By overcoming the permeability barrier of mammalian cell membranes to the sugars, recent studies have clearly demonstrated improved cryosurvival of somatic cells. Therefore, we hypothesize that when present both inside and outside cells, sugars protect oocytes against freezing-induced injury by their excellent glass formation abilities and direct interactions with subcellular components. We also hypothesize that combinations of sugars with conventional penetrating cryoprotectants and development of a novel sugar-based freezing medium will further improve cryosurvival. We will test these hypotheses systematically using intra- and extracellular sugars with different glass transition temperatures and molecular sizes, and then combining the most effective sugars with conventional cryoprotectants. Next, using a coupled mechanistic model of water transport and intracellular ice formation, we will establish an optimal thermodynamic pathway for oocyte freezing in the presence of sugar/cryoprotectant. Finally, we will test developmental capacity of cryopreserved oocytes to term. Our studies will contribute to understanding fundamental aspects of oocyte cryobiology as well as cryoprotective mechanisms of sugars. We predict that the proposed research will result in a reliable preservation technique for primate oocytes by circumventing many of the difficulties associated with conventional protocols.
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