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了解，以及大鼠对新兴基因组学和干细胞生物学领域的重要性，美国国立卫生研究院发起了大鼠基因组计划和大鼠基因组数据库。NIH主办的关于大鼠模型优先事项的会议建议建立一个国家大鼠遗传资源中心，以支持大鼠生殖系改良的研究，包括为大鼠精子冷冻保存的研究提供耳朵标记资金，以缓解储存和运输问题，并改进大鼠的体外受精技术。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，在过去的40年里只发表了两篇论文，试图优化大鼠精子的冷冻保存(Nakatsukasa等人。2001年；2003年)。这项工作确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，或描述该物种的冷冻保存过程。在这项提案中，我们将尝试实现三个非常重要的目标，因为它涉及到精子的冷冻保存，特别是大鼠精子的冷冻保存。首先，我们将试图通过优化冷冻速度来优化大鼠精子的冷冻保存，仔细描述冷冻过程中精子的水传输特性，并优化冷冻保护介质。其次，我们将根据我们对冷冻机制的理解，从分子和结构水平确定冷冻过程对精子造成的损害，并设计和测试将这种损害降至最低的方法。最后，我们将标准化减少损伤和优化冷冻保存结果的方法。这些方法体现在以下3个具体目标上：具体目标1：确定最有效的冷冻保存大鼠精子的处理方法和CPA液。特定目的2：测定冷冻过程中大鼠精子质膜的生物物理水分传输特性。具体目标3：设计和测试保护大鼠精子免受冷冻损伤的策略。

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.