FUNDAMENTAL CRYOBIOLOGY OF MACAQUE SPERMATOZOA

来自非人类灵长类动物的冷冻精子已被用于人工智能和试管受精，但猕猴精子和人类精子一样，根据捐精者的不同，有非常不同的冷冻保护要求。这种生物变异性限制了在开发冷冻保存方案方面的进展，该方案可以在所有精液样本中保留精子的活力。这一重大问题可能会因精子冷冻保护要求的显著差异而加剧，这些要求不受基因改变或独特个体的影响。这项提议的长期目标是确保有价值的人类疾病灵长类模型的保存。主要目标是对猕猴精子的基本冷冻生物学有一个清晰的了解。第二个目标是建立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生化组成和生物物理特性的主要方面，渗透应激诱导的猕猴精子细胞损伤的程度，温度对精子对渗透应激反应的影响，以及确定冷冻保护剂暴露是否可以调节因冷却和渗透应激而产生的不利细胞特性。然后，将根据拟议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。该项目拥有一支高素质的研究团队，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。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被用来评估精子膜的相变。计算机精子活动率分析、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性损伤。然后，将使用数学建模来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.