STUDIES OF SPERM FUNCTION IN MAMMALIAN FERTILIZATION

哺乳动物受精过程中精子功能的研究

• 批准号: 3312954
• 负责人: PATRICIA J OLDS-CLARKE
• 金额: $ 12.17万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-09-01 至 1995-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3312954
• 关键词: acidity /alkalinity; acrosome; calcium; cell membrane; chromosomes; developmental genetics; enzyme inhibitors; fertility promoting drug; fluorescent dye /probe; galactosyltransferases; gene mutation; genes; laboratory mouse; lactalbumin; sperm; sperm capacitation; sperm motility

Very little is known about the molecular mechanisms controlling hyperactivation (HA) and how abnormal movements affect sperm transport through the female genital tract and penetration of the egg investments. The PI has shown that sperm from tw32/+ mice (tw32/+ sperm) have abnormal motility: HA occurs prematurely, and most of the nonhyperactivated motile sperm are abnormally slow. The PI will determine whether precocious HA or slow speed is also characteristic of other t haplotypes (Aim IA). Collaborative studies have shown that the axonemes of tw32/+ sperm are unusually sensitive to calcium (Ca). The PI will determine whether the very slow movements of sperm from mice carrying two t haplotypes are also sensitive, by (1) treating intact sperm with drugs that lower the intracellular free [Ca], and (2) measuring the [Ca] necessary to change flagellar curvature in demembranated sperm (Aim IB). Changes in invertebrate flagellar movement are controlled by cAMP, Ca-binding proteins, or both. Preliminary data suggest that tw32/+ sperm may contain elevated cAMP levels. The PI will determine whether there is a relationship between cAMP and premature HA by (1) measuring cAMP in tw32/+ sperm and (2) perturbing cAMP levels to affect the timing of HA (Aim IIA). Also the Ca-binding proteins of tw32/+ sperm flagella will be characterized by diagonal electrophoresis (Aim IIB). Since the PI has evidence that tw32/+ sperm have normal levels of capacitation, acrosome reaction and zona-binding, their only abnormality relevant to penetration of the investments is in motility. Therefore, the PI will determine whether tw32/+ sperm are abnormal in penetration of the investments by counting the numbers of sperm able to penetrate each investment (Aim II). Experiments from the PI's lab suggest that tw32/+ sperm that do not carry the tw32 haplotype (+ t sperm) are dysfunctional in fertilization in vivo. Although the mechanism of dysfunction is unknown, it must occur between the uterus and the site of gamete interaction and could involve abnormal sperm motility. The Pi will test the hypothesis that the dysfunction of + t sperm occurs in passage through the uterotubal junction by determining whether + t sperm can be recovered from oviductal populations, using the polymerase chain reaction to amplify a sequence which is different for + and t DNA (Aim IV). Determine the site of + t dysfunction will help to determine its nature. These experiments will increase our understanding of how Ca controls hyperactivation and how specific types of movements affect sperm fertilizing ability.

对于控制的分子机制知之甚少 过度激活 (HA) 以及异常运动如何影响精子运输 通过女性生殖道并渗透卵子投资。 PI显示来自tw32/+小鼠的精子（tw32/+精子）有异常 运动性：HA 过早发生，并且大多数非过度活跃的运动性 精子异常缓慢。 PI 将确定 HA 是否早熟 慢速也是其他 t 单倍型的特征 (Aim IA)。 合作研究表明 tw32/+ 精子的轴丝是 对钙 (Ca) 异常敏感。 PI 将决定是否 携带两种 t 单倍型的小鼠的精子运动也非常缓慢 敏感，通过（1）用降低精子活力的药物处理完整的精子 细胞内游离 [Ca]，以及 (2) 测量改变所需的 [Ca] 脱膜精子的鞭毛弯曲（Aim IB）。 变化 无脊椎动物鞭毛运动由 cAMP、Ca 结合控制 蛋白质，或两者兼而有之。 初步数据表明 tw32/+ 精子可能含有 cAMP 水平升高。 PI 将确定是否存在 通过 (1) 测量 tw32/+ 中的 cAMP 来了解 cAMP 和过早 HA 之间的关系 精子和 (2) 扰乱 cAMP 水平以影响 HA 的时间（目标 IIA）。 tw32/+ 精子鞭毛的 Ca 结合蛋白也将被表征 通过对角线电泳（Aim IIB）。 由于 PI 有证据表明 tw32/+ 精子具有正常水平的获能、顶体反应和 透明带结合，它们唯一与穿透有关的异常 投资在于动力。 因此，PI 将确定是否 通过计算 tw32/+ 精子对投资的渗透异常 能够穿透每个投资的精子数量（目标 II）。 实验 PI 实验室表明，不携带 tw32 的 tw32/+ 精子 单倍型（+ t精子）在体内受精功能障碍。 虽然 功能障碍的机制尚不清楚，它一定发生在子宫之间 和配子相互作用的部位，可能涉及异常精子 动力。 Pi 将检验 + t 功能障碍的假设 精子在通过子宫输卵管连接处时发生，通过确定 是否可以使用 + t 精子从输卵管群体中回收 聚合酶链反应放大与 + 不同的序列 和 t DNA（目标 IV）。 确定 +t 功能障碍的部位将有助于 决定其性质。 这些实验将加深我们对 Ca 如何控制过度激活以及特定类型的运动如何影响 精子的受精能力。