BASIC AND APPLIED RESEARCH ON SPERM CRYOPRESERVATION

精子冷冻保存的基础与应用研究

• 批准号: 3327146
• 负责人: JAMES W OVERSTREET
• 金额: $ 11.63万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3327146
• 关键词: acidity /alkalinity; acrosome; artificial fertilization; bioassay; calcium flux; cryopreservation; cryoprotective agents; cyclic AMP; fertilization; freeze etching; hamsters; human tissue; image processing; infrared spectrometry; phase change; sperm; sperm motility

The long range goal of this project is to improve cryopreservation (CP) procedures for human sperm, so that more sperm competent to fertilize eggs will survive frozen storage, and the success rate of artificial insemination will be increased. The effect of different CP protocols on the pattern and vigor of sperm movement will be determined by multivariate, computer aided sperm analysis, and the hypothesis that changes in movement patterns are due to altered internal calcium, pH or cAMP rather than an altered flagellar motor will be tested. The ability of sperm immobilized by CP to be reactivated by experimentally altered internal calcium, pH, or cAMP will be determined. The effect of a variety of CP protocols on acrosomal function of motile sperm will be determined by studying acrosomal structure, acrosin content, maintenance of an acidic acrosomal interior, acrosome stability during in vitro incubation, and the response of sperm to a stimulator of the acrosome reaction. The ability of CP sperm to bind to the zona pellucida and to fuse with the oolemma will be determined. Fourier transform infrared spectroscopy will be used to detect lipid phase transitions in sperm, and the hypothesis that these transitions are a source of damage during CP will be tested by comparing transition temperature (Tm) for sperm that survive CP this the Tm of sperm that do not. Further, agents known to depress Tm in other systems will be tested for their value as sperm cryoprotectants. The hypothesis that phase separations in the plane of the membrane cause redistribution of lipids and proteins will be tested by freeze-fracture. Results of the cell biology studies will be utilized in the context of an empirical testing program which will select a set of new CP methods for further testing with bioassays of sperm function. The choice of bioassays will be based on paired observations of CP sperm exposed to cervical mucus in vitro and in vivo (i.e., recovered after AI). The functional competence of CP sperm will be determined in these assays by their swimming characteristics, their acrosomal integrity, their survival and pattern of movement during incubation under standard in vitro conditions and their ability to undergo the acrosome reaction in response to a biological stimulus. The selected bioassays will be used to identify a superior CP method from among the candidates generated by the empirical testing. Clinical studies will subsequently be carried out in which the superior CP method will be used for AI of study subjects and sperm will be recovered from the subjects for functional testing in vitro.

该项目的长期目标是改善冷冻保存 (CP) 人类精子的程序，使更多的精子有能力受精 卵子能够在冷冻保存中存活，并且人工受精的成功率 授精量将会增加。 不同CP协议的影响 精子运动的模式和活力将由以下因素决定 多变量计算机辅助精子分析，以及假设 运动模式的变化是由于内部钙、pH 值或 将测试 cAMP 而不是改变的鞭毛运动。 能力 被CP固定的精子通过实验改变被重新激活 将测定内部钙、pH 或 cAMP。 的效果 关于活动精子顶体功能的各种 CP 方案将 通过研究顶体结构、顶体素含量、维持情况来确定 酸性顶体内部的影响，体外顶体稳定性 孵化，以及精子对顶体刺激物的反应 反应。 CP 精子与透明带结合并结合的能力 与 oolemma 的融合将被确定。 傅里叶变换红外 光谱学将用于检测精子中的脂质相变，以及 假设这些转变是 CP 期间的损害来源 将通过比较精子的转变温度 (Tm) 进行测试 存活 CP 是不存在的精子的 Tm。 此外，已知代理 在其他系统中抑制 Tm 将测试其作为精子的价值 冷冻保护剂。 平面内相分离的假设 将测试膜引起脂质和蛋白质的重新分布 通过冷冻断裂。 细胞生物学研究的结果将在以下背景下使用： 经验测试程序将选择一套新的 CP 方法 通过精子功能的生物测定进一步测试。 的选择 生物测定将基于对暴露于 CP 精子的配对观察 体外和体内宫颈粘液（即 AI 后恢复）。 这 CP 精子的功能能力将在这些测定中通过以下方式确定 它们的游泳特性、顶体完整性、生存能力 以及标准体外培养过程中的运动模式 条件及其进行顶体反应的能力 到生物刺激。 选定的生物测定将用于 从生成的候选方法中识别出更好的 CP 方法 实证测试。 随后将进行临床研究 高级 CP 方法将用于研究对象的 AI 以及 将从受试者体内回收精子进行功能测试 体外。