PROTOCOLS FOR MATURING AND FREEZING MONKEY OOCYTES

描述（由申请人提供）：本申请的目的是设计生产具有发育能力的体外成熟灵长类动物卵母细胞的方案，并开发NHP卵母细胞的冷冻保存方法。该项目整合了三个区域灵长类动物研究中心（rprc）研究人员的专业知识和资源，以解决这些互补的目标。目前，与体内成熟卵母细胞相比，卵母细胞的IVM成功率明显降低，特别是对未受刺激卵巢的卵母细胞的IVM。最初的实验将使用来自7天卵泡刺激激素（FSH）启动的雌性的卵母细胞，但随着成功率的提高，来自4天卵泡刺激激素启动和非刺激卵巢的卵母细胞将用于实验。IVM方法的改进将包括培养条件的发展，以维持卵丘-卵母细胞的连接，并可逆地抑制核成熟。一旦设计出这些方法，就可以开发出IVM的两步程序。第一步将延迟核成熟，并允许研究提高细胞质成熟的方法。第二步将允许并促进核成熟。在平行实验中，研究人员将继续努力改进成熟和未成熟卵母细胞的冷冻保存方法。该方法的一个关键部分将是确定有机渗透剂和/或其他介质补充剂是否改变卵母细胞对冷损伤或渗透休克的敏感性。随后的实验将推导出冷冻保存的平衡和非平衡（玻璃化）方法。体外受精和低温保存都会对卵母细胞造成损伤，导致胚胎发育能力下降。在改变体外受精和低温保存的程序变得更加成功之前，另一种技术可能为提高这些卵母细胞的发育能力提供一种临时解决方案。将细胞质从体内成熟的、具有发育能力的卵母细胞转移到IVM或冷冻/解冻的卵母细胞可以克服其发育能力下降的问题。一个有效的灵长类动物卵母细胞低温保存和体外受精系统将为早期发育研究提供大量的卵母细胞，并有助于灵长类动物的繁殖和保护工作。

DESCRIPTION (provided by applicant): 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.