Harkema W, Harrison R A, Miller N G, Topper E K, Woelders H
Department of Animal Reproduction, Research Institute for Animal Science and Health, Lelystad, The Netherlands.
Biol Reprod. 1998 Feb;58(2):421-30. doi: 10.1095/biolreprod58.2.421.
In this investigation we sought to determine whether sperm capacitation in vitro is accompanied by changes in the functional presence of zona binding sites on the plasma membrane of boar spermatozoa. During sperm incubation at 39 degrees C in various modifications of a Tyrode's-based in vitro fertilization medium, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins, using a flow cytometer. Propidium iodide was routinely included to allow simultaneous assessment of membrane integrity; rhodamine-conjugated peanut agglutinin was used to assess acrosomal status. During incubation in the fertilization medium, a subpopulation of live acrosome-intact spermatozoa developed enhanced binding of the fluorescein-conjugated solubilized zona proteins. Microscopy revealed that the increase in cytometrically detected zona binding was paralleled by an increase in the area on the sperm head to which zona proteins bound, from the apical region to the whole of the acrosomal region. The changes were accelerated by phosphodiesterase inhibitors, were attenuated by omission of bicarbonate, and were completely inhibited by addition of EGTA. In the fertilization medium, numbers of sperm showing enhanced zona binding maximized after 60-90 min. This time course is somewhat similar to that reported by others for development of egg-penetrating ability in vitro. We suggest that the observed changes in zona binding ability bring about optimal sperm-egg attachment; they may also relate to induction of the acrosome reaction by zona pellucida components. In consequence, the zona binding changes may be an important part of the process by which the sperm acquires fertilizing ability as a result of capacitation.
在本研究中，我们试图确定公猪精子体外获能过程中，其质膜上透明带结合位点的功能存在是否会发生变化。在基于台氏液的体外受精培养基的各种改良配方中，将精子于39℃孵育，使用流式细胞仪，通过荧光素偶联的可溶性透明带蛋白评估单个精子的透明带结合能力。常规加入碘化丙啶以同时评估膜完整性；使用罗丹明偶联的花生凝集素评估顶体状态。在受精培养基中孵育期间，一群存活且顶体完整的精子对荧光素偶联的可溶性透明带蛋白的结合能力增强。显微镜检查显示，流式细胞术检测到的透明带结合增加与透明带蛋白结合的精子头部区域从顶端区域增加到整个顶体区域平行。磷酸二酯酶抑制剂可加速这些变化，去除碳酸氢盐可减弱这些变化，加入乙二醇双乙胺醚四乙酸（EGTA）可完全抑制这些变化。在受精培养基中，显示增强的透明带结合的精子数量在60 - 90分钟后达到最大值。这个时间进程与其他人报道的体外卵穿透能力发展的时间进程有些相似。我们认为，观察到的透明带结合能力的变化导致了最佳的精卵附着；它们也可能与透明带成分诱导顶体反应有关。因此，透明带结合变化可能是精子因获能而获得受精能力过程的一个重要部分。
