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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在调节精子获能过程中的功能表征，以及3：调节精子头对头凝集的信号级联。实验 1 和 3 取得了显着的结果，并在此进行总结。实验1揭示了在旨在促进体外获能的培养过程中与精子结合的抗凝集素的变化。通过蛋白质印迹光密度分析评估，精子结合的抗凝集素的相对量在第一个 45 分钟孵育后几乎减半，然后逐渐下降。免疫细胞化学观察表明，许多精子在孵化过程中精子结合抗凝集素的减少主要发生在顶体。这些结果表明，与精子顶体结合的大部分抗凝集素在体外获能过程的早期阶段被释放。实验3旨在揭示碳酸氢盐-腺苷酸环化酶（AC）-cAMP-蛋白激酶（PK）系统在精子头对头凝集中的作用。在补充有 100 μM forskolin（一种 AC 激活剂）的无 Ca mKRB 中孵育的样本中，与对照样本相比，在顶体处相互凝集的精子百分比明显更高。添加二丁酰 CAMP（dbcAMP，一种 CAMP 依赖性 PK 激活剂：PKA）代替毛喉素，在 1 至 1,000 μM 之间以剂量依赖性方式增加头对头凝集精子的百分比。然而，添加 dbcAMP (1,000 μM) 的效果因 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系统介导导致精子头对头凝集的信号通路。较少的