Ashworth P J, Harrison R A, Miller N G, Plummer J M, Watson P F
Department of Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom.
Mol Reprod Dev. 1995 Feb;40(2):164-76. doi: 10.1002/mrd.1080400205.
Boar and ram spermatozoa were incubated in Tyrode's medium in the presence or absence of bicarbonate/CO2, a component believed essential for capacitation. At intervals, samples were stained with a range of FITC-lectins to detect changes in surface glycoconjugates, using a rapid staining technique to avoid problems of lectin toxicity. The samples were then analysed directly by flow cytometry, using propidium iodide to distinguish dead cells. In the presence of bicarbonate, a live subpopulation of spermatozoa developed, which in both animal species showed higher binding affinities towards Phaseolus Vulgaris Agglutinin (PHA-E), Sophora Japonica Agglutinin (SJA), and Soybean Agglutinin (SBA), and lower binding affinity towards Erythrina Cristagalli Lectin (ECL). In boar samples, the modified subpopulation reached a maximum after 3 hr incubation, whereas in ram samples it maximized after 1.5 hr. No changes were seen when spermatozoa were incubated in bicarbonate-free medium. The bicarbonate-induced changes in lectin binding were not due to the onset of acrosome reactions, because spermatozoa induced to undergo acrosome reactions with the ionophore A23187 displayed very different lectin-binding patterns. Tested on boar spermatozoa, seminal plasma not only inhibited but reversed the bicarbonate-induced development of the modified subpopulation. EGTA also inhibited development of boar sperm subpopulations; excess Ca2+ was unable to overcome this inhibition, suggesting that multivalent metal ions might be involved in bicarbonate's action. We conclude that bicarbonate causes a loss of surface coating material with affinity for ECL and an unmasking of binding sites for SBA, SJA and PHA-E. A modified subpopulation of live spermatozoa is thereby established, which appears to maximize at a rate in accord with reported capacitation times.
将公猪和公羊的精子在含有或不含碳酸氢盐/二氧化碳的泰罗德氏培养基中孵育，碳酸氢盐/二氧化碳被认为是精子获能所必需的成分。每隔一段时间，使用一系列异硫氰酸荧光素（FITC）标记的凝集素对样本进行染色，以检测表面糖缀合物的变化，采用快速染色技术以避免凝集素毒性问题。然后使用碘化丙啶区分死细胞，通过流式细胞术直接对样本进行分析。在有碳酸氢盐存在的情况下，出现了一个活精子亚群，在这两种动物中，该亚群对菜豆凝集素（PHA-E）、槐凝集素（SJA）和大豆凝集素（SBA）表现出更高的结合亲和力，而对刺桐凝集素（ECL）的结合亲和力较低。在公猪样本中，经过3小时孵育后，修饰后的亚群达到最大值，而在公羊样本中，1.5小时后达到最大值。当精子在不含碳酸氢盐的培养基中孵育时，未观察到变化。碳酸氢盐诱导的凝集素结合变化并非由于顶体反应的开始，因为用离子载体A23187诱导发生顶体反应的精子表现出非常不同的凝集素结合模式。对公猪精子进行测试时，精浆不仅抑制了修饰后亚群的形成，而且使其逆转。乙二醇双乙胺四乙酸（EGTA）也抑制了公猪精子亚群的形成；过量的钙离子无法克服这种抑制作用，这表明多价金属离子可能参与了碳酸氢盐的作用。我们得出结论，碳酸氢盐导致对ECL具有亲和力的表面涂层物质丢失，并使SBA、SJA和PHA-E的结合位点暴露。由此建立了一个活精子的修饰亚群，其似乎以与报道的获能时间一致的速率达到最大值。
