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

本研究进行了以下三个实验：1. 精子结合抗凝集素在获能过程中的变化，2：精浆中抗凝集素的分离及其在精子获能调控中的功能表征，3：调节精子头对头凝集的信号级联。在实验1和实验3中得到了重要的结果，在这里进行总结。实验1揭示了与精子结合的抗凝集素在体外培养过程中的变化，旨在促进体外获能。通过免疫印迹-密度分析评估，精子结合抗凝集素的相对量在第一个45分钟孵育后几乎减半，随后逐渐下降。免疫细胞化学观察表明，在孵育过程中，许多精子结合抗凝集素的降低主要发生在顶体上。这些结果表明，在体外获能过程的早期阶段，大量与精子顶体结合的抗凝集素被释放出来。实验3揭示了碳酸氢盐-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。在添加100 μM forskolin（一种AC激活剂）的无ca mKRB中培养的样品中，与对照样品相比，精子在顶体处相互凝集的比例显著提高。添加二丁基CAMP （dbcAMP，一种CAMP依赖性PK: PKA的激活剂）代替福斯olin，在1 ~ 1000 μM范围内以剂量依赖性的方式增加了头对头凝集精子的百分比。然而，添加dbcAMP （1000 μM）的效果被Rp-cAMPS （1000 μM， PKA抑制剂）或H-89 （5 μM， PKA抑制剂）所减弱。这些结果表明，碳酸氢盐- ac - camp - pk系统介导了一个导致精子头对头凝集的信号通路。少

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