Changes and functions of Anti-Agglutinin in boar spermatozoa during their capacitation

公猪精子获能过程中抗凝集素的变化及功能

基本信息

批准号：
11460128
负责人：
KATO Seishiro
金额：
$ 4.67万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

Three following experiments have been undertaken in this study ; 1. changes of sperm-bound anti-agglutinin during the capacitation, 2 : isolation of anti-agglutinin from seminal plasma and functional characterization of this protein on the regulation of sperm capacitation, and 3 : signaling cascade regulating sperm head-to-head agglutination. Significant results have been obtained in Experiments 1 and 3 and summarized here. Experiment 1 has revealed changes in anti-agglutinin bound to spermatozoa during the incubation designed to promote the capacitation in vitro. The relative amount of sperm-bound anti-agglutinin, assessed by Western blotting-densitometric analyses, was almost halved after the first 45-min incubation and then decreased gradually thereafter. Immunocytocheinical observation showed that the decrease of the sperm-bound anti-agglutinin occurred mainly on acrosomes in many spermatozoa during the incubation. These results indicate that a large portion of the anti-agglutinin … More bound to sperm acrosomes is released at an early stage of the Capacitation process in vitro. Experiment 3 has been conducted to reveal roles of bicarbonate-adenylyl cyclase (AC)-cAMP-protein kinase (PK) system in sperm head-to-head agglutination. In the samples incubated in Ca-free mKRB supplemented with 100 μM forskolin (an AC activator), significantly higher percentages of the spermatozoa were agglutinated with one another at the acrosome, as compared with control samples. Addition of dibutyryl CAMP (dbcAMP, an activator of CAMP-dependent PK : PKA), instead of forskolin, increased the percentages of head-to-head agglutinated spermatozoa in a dose-dependent manner between 1 and 1,000 μM. However, the effects of the addition of dbcAMP (1,000 μM) were attenuated by treatments with Rp-cAMPS (1,000 μM, a PKA inhibitor) or H-89 (5 μM, a PKA inhibitor). These results indicate that bicarbonate-AC-cAMP-PK system mediates a signaling pathway leading to head-to-head agglutination of spermatozoa. Less

本研究进行了以下三个实验；1.获能过程中精子结合的抗凝集素的变化，2：从精浆中分离抗凝集素以及该蛋白质对精子获能调节的功能表征，以及3：信号级联调节精子头对头凝集已在实验 1 和 3 中获得，实验 1 揭示了结合的抗凝集素的变化。免疫细胞化学观察显示，通过蛋白质印迹光密度分析评估，在培养过程中促进精子获能的精子结合抗凝集素的相对量在第一个45分钟培养后几乎减半，然后逐渐减少。精子结合抗凝集素的减少主要发生在许多精子的孵化过程中的顶体上。与精子顶体结合的抗凝集素部分在体外获能过程的早期阶段被释放，以揭示碳酸氢盐-腺苷酸环化酶 (AC)-cAMP-蛋白激酶 (PK) 系统的作用。在补充有 100 μM forskolin（AC 激活剂）的无 Ca mKRB 中孵育的样本中，精子头对头凝集的百分比显着提高。与对照样品相比，添加二丁酰 CAMP（dbcAMP，一种 CAMP 依赖性 PK 激活剂：PKA）而不是毛喉素，精子在顶体处相互凝集，增加了头对头凝集精子的百分比。 1 至 1,000 μM 之间存在剂量依赖性，但是添加 dbcAMP 的效果会受到影响。 (1,000 μM) 通过 Rp-cAMPS（1,000 μM，PKA 抑制剂）或 H-89（5 μM，PKA 抑制剂）处理而减弱。这些结果表明碳酸氢盐-AC-cAMP-PK 系统介导了一条信号通路。精子的头对头凝集较少。