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

本研究进行了三个实验：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对精子获能的调节作用；3.信号通路调节精子头对头的凝集。在实验1和实验3中取得了重要结果，并在此进行了总结。实验1揭示了在体外促进获能的孵育过程中，精子结合的抗凝集素的变化。通过Western blotting-密度分析评估，精子结合抗凝集素的相对量在最初的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，在孵育过程中，精子结合抗凝素的减少主要发生在许多精子的顶体上。这些结果表明，很大一部分抗凝集素…更多与精子顶体结合的物质在体外获能过程的早期阶段被释放。实验3揭示了碳酸氢钠-腺苷环化酶(AC)-cAMP-蛋白激酶(PK)系统在精子头对头凝集中的作用。在无钙mKRB+100μM Forskolin(AC激活剂)中孵育的样品中，精子在顶体相互凝集的百分比显著高于对照样品。在1~1,000μM范围内，加入二丁酰cAMP(cAMP，cAMP依赖的PK激活剂：pKA)而不是Forsklin，可剂量依赖性地增加精子头对头凝集精子的百分率。然而，加入dBcAMP(1,000μM)的作用可被RP-cAMP(1,000μM，PKA抑制剂)或H-89(5μM，PKA抑制剂)所减弱。这些结果表明，碳酸氢钠-AC-cAMP-PK系统介导了导致精子头对头凝集的信号通路。较少

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