New Approaches to Rat Sperm Cryopreservation

描述（由申请人提供）：鉴于大鼠模型系统为我们提供了对人类疾病生物学的基本理解，以及大鼠对基因组学和干细胞生物学新兴领域的重要性，NIH启动了大鼠基因组计划和大鼠基因组数据库。美国国立卫生研究院主办的关于大鼠模型优先事项的会议建议建立国家大鼠遗传资源中心，以支持大鼠生殖系修饰的研究，包括为大鼠精子冷冻保存研究提供专项资金，以减轻储存和运输问题，并改善大鼠体外受精技术。不幸的是，目前大鼠精子冷冻保存的技术水平非常糟糕。对文献的电子搜索显示，过去40年来只有两篇旨在优化大鼠精子冷冻保存的论文（Nakatsukasa等人，2001; 2003）。这项工作建立了大鼠精子冷冻保存的可行性，但没有试图优化技术，了解冷冻保存对精子造成的损害，或表征该物种的冷冻保存过程。在这个建议中，我们将试图实现三个非常重要的目标，因为它涉及到精子冷冻保存一般和大鼠精子冷冻保存特别是。首先，我们将尝试优化大鼠精子冷冻保存的冷冻速率的基础上仔细表征的水运输性能的精子在冷冻和冷冻保护介质的优化。其次，我们将确定冷冻保存过程对精子在分子和结构水平上造成的损害，并根据我们对机制的理解设计和测试方法以最大限度地减少这种损害。最后，我们将标准化的方法，以减少损伤和优化冷冻保存的结果。这些方法反映在以下3个具体目标中：具体目标1：确定对大鼠精子冷冻保存最有效的处理方法和CPA培养基。 具体目标2：确定冷冻过程中大鼠精子质膜的生物物理水转运特性。 具体目标3：设计和测试策略，以保护大鼠精子免受冷冻保存引起的损害。

DESCRIPTION (provided by applicant): In light of the benefit that the rat model system have provided to our basic understanding of the biology of human disease, and the importance of the rat to emerging fields of genomics and stem cell biology, the NIH initiated the Rat Genome Program and the Rat Genome Database. The NIH sponsored meeting on Rat Model Priorities recommended that a National Rat Genetic Resource Center be established to support research on germ-line modifications in the rat, including ear-marked funding for the study of rat sperm cryopreservation to alleviate the storage and transportation problems, and to improve in vitro fertilization techniques in the rat. Unfortunately, the current state of the art in rat sperm cryopreservation is abysmal. An electronic search of the literature reveals only two papers published in the last 40 years that seeks to optimize the cryopreservation of rat sperm (Nakatsukasa et al. 2001; 2003). This work establishes the feasibility of rat sperm cryopreservation but makes no attempt to optimize the technology, understand the damage caused to the sperm by cryopreservation, or to characterize the cryopreservation process in this species. In this proposal, we will attempt to achieve three very important goals as it relates to sperm cryopreservation in general and rat sperm cryopreservation in particular. First, we will attempt to optimize rat sperm cryopreservation by optimizing the freezing rate based careful characterization of the water transport properties of the sperm during freezing and optimization of the cryoprotective media. Second, we will determine the damage done to the sperm, at the molecular and structural level, caused by cryopreservation process, and design and test methods to minimize this damage based on our understanding of the mechanism. Finally, we will standardize approaches to reduce injury and optimize cryopreservation outcomes. These approaches are reflected in the following 3 specific aims: Specific Aim 1: Determine the handling methods and CPA media most effective for rat sperm cryopreservation. Specific Aim 2: Determine the biophysical water transport properties of rat sperm plasma membrane during freezing. Specific Aim 3: Design and test strategies for protecting rat sperm from damage incurred by cryopreservation.