FUNDAMENTAL CRYOBIOLOGY OF MACAQUE SPERMATOZOA

冷冻保存的非人类灵长类动物精子已被用于人工智能和体外受精，但猕猴精子和人类精子一样，其冷冻保护要求因人而异，具体取决于精子捐赠者。这种生物变异限制了在所有精液样本中保存精子活力的冷冻保存方案的发展。这一重大问题可能会因来自基因改变或独特个体的精子的冷冻保护要求的显著差异而加剧。这项提议的长期目标是确保保存有价值的人类疾病灵长类动物模型。主要目的是对猕猴精子的基本低温生物学有一个清晰的认识。第二个目标是开发一种合理可靠的猕猴精子冷冻保存方法。该项目的具体目标是确定猕猴精子的主要生化组成和生物物理特性，渗透胁迫诱导的猕猴精子细胞损伤程度，温度对精子对渗透胁迫反应的影响，以及确定低温保护剂暴露是否可以调节冷却和渗透胁迫导致的不良细胞特性。然后，根据所提出的实验产生的猕猴精子的基本知识，合理开发优化的冷冻保存方案。该项目拥有一支由加州大学戴维斯分校和明尼苏达大学的细胞生物学家和低温生物学家组成的高素质研究团队。该项目将利用多种技术来实现这些目标。傅里叶变换红外光谱将用于评估精子膜相变。计算机精子活力分析、荧光和共聚焦显微镜、流式细胞术、差示扫描量热法和细胞体积分析将用于评估精子功能参数和对猕猴精子的亚致死损伤。数学建模将用于构建优化的低温保存方案。

Cryopreserved sperm from non-human primates have been used for AI and IVF, but macaque sperm, like human sperm, have highly variable cryoprotection requirements depending on the individual sperm donor. This biological variability has restricted progress in developing a cryopreservation protocol that preserves sperm motility in all semen samples. This significant problem is likely to be exacerbated by significant variability in the cryoprotection requirements of sperm from genetically altered or unique individuals. The long-range goal of this proposal is to assure the preservation of valuable primate models of human disease. The principal objective is to develop a clear understanding of the fundamental cryobiology of macaque sperm. The second objective is to develop a rational and reliable method for cryopreservation of macaque sperm. The specific goals of this project are to determine the major aspects of biochemical composition and biophysical characteristics of macaque sperm, the extent of osmotic stress-induced cell damage in macaque sperm, the effects from temperature on spermatozoal responses to osmotic stress, and to determine whether cryoprotectant exposure can modulate adverse cellular properties resulting from cooling and osmotic stress. Rational development of an optimized cryopreservation protocol will then be developed based on the fundamental knowledge of macaque sperm generated from the proposed experiments. This project has a highly qualified research team comprised of cell biologists and cryobiologists from the University of California, Davis and the University of Minnesota. The project will utilize numerous techniques to accomplish these goals. Fourier-transform infrared spectroscopy will be used to assess sperm membrane phase transitions. Computerized sperm motility analysis, epifluorescence and confocal microscopy, flow cytometry, differential scanning calorimetry, and cell volumetric analysis will be used to evaluate sperm functional parameters and sublethal damage to macaque sperm. Mathematical modeling will then be used to construct optimized cryopreservation protocols.