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

描述(由申请人提供)：有效保存猕猴(Macaca Mulatta)的遗传资源对国家研究资源中心的使命至关重要。这一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。非人灵长类动物，特别是基因独特的猕猴，是人类疾病的重要模型，应用于不孕不育和避孕、传染病和疫苗开发、药物和酒精成瘾、神经疾病和再生医学。与人类精子相似，来自NHP的冷冻精子显示出不同的冷冻保护成功率，这取决于不同的精子捐赠者。捐精者通常是根据冷冻成功的结果来选择的，然而，许多个体男性，包括研究项目中的恒河猴，精液质量无法在冷冻后存活下来。对于胚胎干细胞产生的NHP，体细胞核移植，以及希望繁殖特定个体的基因的转基因，这一点可能尤其正确。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，了解精子中与冷冻保存相关的生理反应将有助于提高精子的冷冻存活率。我们的长期目标是了解精子冷冻保存的关键机制，以便以最佳方式保存来自各种基因独特和有价值的NHP男性的精子。我们的方法是在细胞生理水平上研究渗透应激和氧化应激的机制，包括膜、细胞信号和亚细胞功能的评估，使用荧光显微镜、流式细胞仪、冷冻显微镜和蛋白质组学方法。作为一个由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生物学方面的经验相结合，我们能够很好地开展这项拟议的研究。

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.