The objective of this project is to devise technical protocols for in vitro maturation (IVM) and cryopreservation of non-human primate oocytes using rhesus monkeys. At present, IVM success in primates is very poor compared with laboratory species such as cattle. Different culture methods for supporting primate IVM will be tested to find an optimal protocol. IVM protocols will be empirically determined by testing the addition of specific amino acids, energy substrates and growth factors, which has proved successful with oocytes of other laboratory species. A key part of this approach will be use of protein-free culture media to avoid unknown factors associated with serum or serum albumin preparations, leading to development of more reproducible protocols. In a parallel study, the ability of oocytes to survive a range of osmotic shocks, cooling rates and types of cryoprotective agents will be measured. This information will be used to construct new cryopreservation protocols. The success of both IVM and cryopreservation protocols will be measured by morphological and developmental criteria, including determination of the integrity of metaphase II spindle structure, cortical granules and zonae pellucidae; examination of the organization of oocyte cytoplasm (e.g., mitochondrial distribution) using confocal and multiphoton microscopy; and assessment of viability of oocytes by their ability to undergo fertilization and embryo development, using standard protocols. Embryo development will be further evaluated by timelapse videomicrography and by embryo transfers to validate normality. Then, cryopreserved oocytes will be used to test the ability of the optimized IVM protocol to support maturation of stressed oocytes. An effective system of primate oocyte cryopreservation and IVM would provide a means for producing and making available large numbers of primate embryos for other studies on early development and would also assist primate breeding and conservation efforts.
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