BASIC AND APPLIED RESEARCH ON SPERM CRYOPRESERVATION

精子冷冻保存的基础与应用研究

• 批准号: 3327144
• 负责人: JAMES W OVERSTREET
• 金额: $ 17.11万
• 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/3327144
• 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.

该项目的远距离目标是改善冷冻保存（CP） 人类精子的程序，因此有能力施肥的精子 鸡蛋将在冷冻存储中幸存下来，人工的成功率 授精会增加。 不同CP协议对 精子运动的模式和活力将由 多变量，计算机辅助精子分析和假设 运动模式的变化是由于内部钙，pH或 将测试营地而不是改变的鞭毛电动机。 能力 由CP固定的精子被实验改变重新激活 将确定内部钙，pH或营地。 一个 关于运动精子的杂色症功能的各种CP方案将是 通过研究耕种结构，阿素蛋白含量，维护来确定 在体外的酸性顶体内部，邻属体稳定性 孵育，精子对Acrosom的刺激器的反应 反应。 CP精子与Zona pellucida结合的能力和 将确定与卵形的融合。 傅立叶变换红外 光谱法将用于检测精子中的脂质相跃迁，并且 这些过渡是CP期间损害的根源的假设 将通过比较精子的过渡温度（TM）来测试 在CP中幸存下来，这是无精子的TM。 此外，已知的代理 其他系统中的凹陷TM作为精子的价值将进行测试 冷冻保护剂。 假设相位在平面中的相位 膜导致脂质和蛋白质的重新分布 通过冻结。 细胞生物学研究的结果将在 经验测试程序将选择一组新的CP方法 用精子功能的生物测定进一步测试。 选择 生物测定将基于暴露于CP精子的配对观察结果 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确定 他们的游泳特征，耕种完整性，生存 在标准体外孵育过程中的运动模式 条件及其对响应进行选择性反应的能力 进行生物刺激。 选定的生物测定将用于 从由 经验测试。 随后将在 哪种CP方法将用于研究对象的AI，并且 精子将从受试者中回收以进行功能测试 体外。