The objectives of this application are to devise protocols for the production of developmentally competent in vitro matured primate oocytes and to develop methods for cryopreservation of NHP oocytes. This project integrates the expertise and resources of investigators at three Regional Primate Research Centers (RPRCs) to address these complimentary objectives. At present, the success rate for IVM of oocytes is markedly reduced compared to in vivo matured oocytes, especially for IVM of oocytes from non-stimulated ovaries. Initial experiments will utilize oocytes from seven-day follicle stimulated hormone (FSH)-primed females, but as success improves, oocytes from four-day FSH-primed and non-stimulated ovaries will be used in experiments. Improvement of IVM methods will include the development of culture conditions that maintain the cumulus-oocyte connections and reversibly inhibit nuclear maturation. Once these methods are devised, a two-step procedure for IVM can be developed. The first step will delay nuclear maturation and allow the study of methods to improve cytoplasmic maturation. The second step will allow and promote nuclear maturation. In parallel experiments, the investigators will continue with efforts to improve methods for cryopreservation of mature and immature oocytes. A key part of the approach will be to determine whether organic osmolytes and/or other media supplements alter the sensitivity of oocytes to chilling injury or osmotic shock. Subsequent experiments will derive equilibrium and non-equilibrium (vitrification) methods for cryopreservation. Both IVM and cryopreservation can cause damage to oocytes, which results in decreased developmental competence of the embryos. Until alterations in the procedures for IVM and cryopreservation become more successful, another technique may provide an interim solution for improving the developmental competence of these oocytes. Transfer of cytoplasm from in vivo matured, developmentally competent oocytes to IVM or frozen/thawed oocytes may overcome their reduced developmental competence. An effective system of primate oocyte cryopreservation and IVM would provide a means to make large numbers of oocytes available for studies on early development and would assist primate breeding and conservation efforts.
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