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对人类疾病生物学的基本理解提供的好处，以及大鼠对基因组学和干细胞生物学新兴领域的重要性，美国国立卫生研究院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大鼠模型优先会议建议建立一个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，并改进大鼠体外受精技术。不幸的是，目前的大鼠精子冷冻保存技术非常糟糕。对文献的电子检索显示，在过去40年里，只有两篇论文发表，旨在优化大鼠精子的冷冻保存（Nakatsukasa et al. 2001; 2003）。本研究确定了大鼠精子低温保存的可行性，但没有尝试优化技术，了解低温保存对精子的损害，或表征该物种的低温保存过程。在本提案中，我们将尝试实现三个非常重要的目标，因为它涉及到精子冷冻保存，特别是大鼠精子冷冻保存。首先，我们将尝试通过优化冷冻速率来优化大鼠精子的冷冻保存，这是基于对精子在冷冻过程中水分运输特性的仔细表征和冷冻保护介质的优化。其次，我们将在分子和结构水平上确定冷冻过程对精子造成的损害，并根据我们对机制的理解设计和测试方法，以尽量减少这种损害。最后，我们将规范减少损伤和优化冷冻保存结果的方法。具体目标1：确定大鼠精子冷冻保存最有效的处理方法和CPA培养基。专项目的2：测定大鼠精子质膜在冷冻过程中的生物物理运水特性。具体目标3：设计和测试保护大鼠精子免受低温保存损伤的策略。

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.