PROTOCOLS FOR MATURING AND FREEZING MONKEY OOCYTES

猴卵母细胞成熟和冷冻方案

• 批准号: 6545363
• 负责人: CATHERINE A VANDEVOORT
• 金额: $ 53.77万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6545363
• 关键词: Macaca mulatta; cell cycle; cryopreservation; cytoplasm; early embryonic stage; egg /ovum; embryo /fetus culture; embryo /fetus tissue transplantation; embryogenesis; epidermal growth factor; fertilization; fluorescence microscopy; follicle stimulating hormone; growth media; immunocytochemistry; insulinlike growth factor; method development; osmotic pressure; polymerase chain reaction; temperature; western blottings

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.

描述（由申请人提供）：本申请的目的是设计用于生产具有发育能力的体外成熟灵长类卵母细胞的方案并开发用于冷冻保存NHP卵母细胞的方法。 该项目整合了三个区域灵长类研究中心 (RPRC) 研究人员的专业知识和资源，以实现这些互补目标。 目前，与体内成熟卵母细胞相比，卵母细胞IVM的成功率明显降低，尤其是来自非刺激卵巢的卵母细胞的IVM。 最初的实验将使用来自经过 7 天卵泡刺激素 (FSH) 刺激的雌性的卵母细胞，但随着成功率的提高，来自经过四天 FSH 刺激和未刺激的卵巢的卵母细胞将用于实验。 IVM 方法的改进将包括开发维持卵丘-卵母细胞连接并可逆地抑制核成熟的培养条件。 一旦设计出这些方法，就可以开发 IVM 的两步程序。 第一步将延迟核成熟并允许研究改善细胞质成熟的方法。 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是确定有机渗透剂和/或其他培养基补充剂是否会改变卵母细胞对冷损伤或渗透压休克的敏感性。 随后的实验将推导出平衡和非平衡（玻璃化）冷冻保存方法。 IVM 和冷冻保存都会对卵母细胞造成损害，从而导致胚胎发育能力下降。 在 IVM 和冷冻保存程序的改变变得更加成功之前，另一种技术可能为提高这些卵母细胞的发育能力提供临时解决方案。 将细胞质从体内成熟、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。 有效的灵长类卵母细胞冷冻保存和 IVM 系统将提供一种手段，使大量卵母细胞可用于早期发育研究，并有助于灵长类动物的育种和保护工作。