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

这项研究已经进行了三个以下实验。 1。在电容期间精子结合的抗吞丁物素的变化，2：从半血压中隔离抗凝集素，该蛋白在调节精子电容的调节中的功能表征，以及3：信号级联反应调节精子头对头凝集。在实验1和3中获得了显着的结果，并在此总结了。实验1揭示了在旨在促进体外电容的孵育过程中与精子结合的抗凝集素的变化。通过蛋白质印迹 - 敏感分析评估的精子结合的抗吞丁物素的相对量几乎减半，然后逐渐增加。免疫细胞化学观察结果表明，在孵育期间，许多精子中，精子抗凝集素的降低主要发生在许多精子中。这些结果表明，在体外的电容过程的早期阶段释放了与精子杂色的大部分抗凝集素…更与精子缩小的结合。已经进行了实验3，以揭示碳酸氢盐辅助环酶（AC） - 碳酸酯 - 蛋白酶激酶（PK）系统在精子正面凝集中的作用。在补充了100μM福克蛋白（AC激活剂）的无CA孵育的样品中，与对照样品相比，精子的百分比显着更高。添加二培养基营地（DBCAMP是CAMP依赖性PK：PKA）而不是Forskolin，以1至1,000μm的剂量依赖性方式增加了头对头凝集的精子的百分比。但是，通过用RP-cAMP（1,000μM）μm，PKA抑制剂）或H-89（5μM，PKA抑制剂）处理添加DBCAMP（1,000μM）的影响。这些结果表明，碳酸氢盐-AC-camp-PK系统介导了信号通路，导致精子的头对头凝集。较少的