Response of Rhesus Sperm to Freezing: Recovery from Osmotic and Oxidative Stress

描述（由申请人提供）：有效保存恒河猴（Macaca mulatta）的遗传资源对国家研究资源中心的使命至关重要。该应用旨在开发高效的猕猴精子冷冻保存，并将有助于通过人工授精和体外受精计划成功繁殖种质资源。非人类灵长类动物（NHPs），特别是遗传独特的猕猴，是人类疾病的重要模型，在不育和避孕、传染病和疫苗开发、药物和酒精成瘾、神经系统疾病和再生医学等方面都有应用。与人类精子类似，来自NHPs的冷冻保存精子显示出不同的冷冻保护成功，这取决于单个精子供体。精子捐献者通常是根据成功的冷冻保存结果来选择的，然而，许多雄性个体，包括研究项目中的恒河猴，其精液质量无法在冷冻保存中存活。这对于从胚胎干细胞、体细胞核移植和转基因中产生的NHPs来说可能尤其正确，因为转基因需要繁殖特定个体的基因型。这项资助分析了一些可能导致低温性能差的机制，并重点研究了统一低温保存这些双峰种群的方法。我们的中心假设是，对精子中与冷冻保存相关的生理反应的理解将导致精子冷冻存活的改善。我们的长期目标是了解精子冷冻保存的关键机制，以便最佳地保存来自各种基因典型独特和有价值的NHP雄性的精子。我们的方法将是在细胞生理学水平上研究渗透和氧化细胞应激的机制，包括膜，细胞信号传导，以及使用荧光显微镜，流式细胞术，低温显微镜和蛋白质组学方法对精子功能进行亚细胞评估。作为一个由来自加州大学戴维斯分校、印第安纳大学/普渡大学和纽卡斯尔大学（澳大利亚）的成员组成的团队，由于我们在基础低温生物学和精子细胞生物学方面的综合经验，我们完全有能力承担这项拟议的研究。

DESCRIPTION (provided by applicant): Efficient preservation of genetic resources of the rhesus monkey (Macaca mulatta) is critical to the mission of the National Center for Research Resources. This application is aimed at the development of efficient macaque sperm cryopreservation and will aid in successful propagation of germplasm through artificial insemination and in vitro fertilization programs. Non-human primates (NHPs), especially genetically unique macaques, are important models of human disease with applications to infertility and contraception, infectious diseases and vaccine development, drug and alcohol addiction, neurological disorders, and regenerative medicine. Similar to human sperm, cryopreserved sperm from NHPs show variable cryoprotection success depending on the individual sperm donor. Semen donors are often selected based on successful cryopreservation results, however, many individual males, including rhesus males in research programs, have semen quality that fails to survive cryopreservation. This is likely to be particularly true concerning NHPs generated from embryonic stem cells, somatic cell nuclear transfer, and transgenics where propagation of a specific individual's genotype is desired. This grant analyzes some of the mechanisms that may cause poor cryoperformance and focuses on methods to uniformly cryopreserve these bimodal populations. Our central hypothesis is that an understanding of cryopreservation-associated physiological responses in sperm will lead to improvements in sperm cryosurvival. Our long-term goal is to develop an understanding of crucial mechanisms involved in cryopreservation of sperm in order to optimally preserve sperm from a variety of genotypically unique and valuable NHP males. Our approach will be to investigate the mechanisms of osmotic and oxidative cellular stress at the cell physiology level which includes membrane, cell signaling, and subcellular assessment of sperm function using fluorescence microscopy, flow cytometry, cryomicroscopy, and proteomics methods. As a team with members from UC Davis, Indiana University/Purdue University, and the University of Newcastle (Australia), we are well positioned to undertake the proposed research, because of our combined experience with fundamental cryobiology and sperm cell biology.