2 SIMPLE METHODS FOR DETECTING ACROSOME-REACTED HUMAN-SPERM
2 SIMPLE METHODS FOR DETECTING ACROSOME-REACTED HUMAN-SPERM
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DOI：
10.1002/mrd.1120150303
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发表时间：
1986-01-01
期刊：
影响因子：
--
通讯作者：
HANSON, FW
中科院分区：
文献类型：
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作者：
CROSS, NL;MORALES, P;HANSON, FW
我们描述了两种在光学显微镜水平上检测人类精子顶体反应的方法。这些技术包括使用超活体染色来检测死亡精子，以区分“生理性”和“变性”顶体反应。将精子与活细胞染色剂Hoechst 33258（一种具有有限膜渗透性的荧光DNA结合染料）一起孵育，洗涤，悬浮于95%乙醇中进行固定和透化，并在载玻片上干燥。然后用兔抗人精子抗血清或荧光标记的豌豆凝集素（PSA）通过间接免疫荧光标记精子。这两种探针强烈标记顶体完整精子的顶体区域。电子显微镜显示PSA结合的主要部位是顶体内容物。顶体反应精子的两种探针的顶体标记均减少;核被Hoechst染色标记的精子被视为无活力，并从试验中排除。这两种检测方法都是快速的，得到类似的结果，并检测到暴露于离子载体A23187后顶体反应的增加。
We describe two methods for detecting acrosome reactions of human sperm at the light microscopic level. The techniques include the use of a supravital stain to detect dead sperm in order to differentiate between "physiological" and "degenerative" acrosome reactions. Sperm are incubated with the supravital stain Hoechst 33258 (a fluorescent DNA-binding dye with limited membrane permeability), washed, suspended in 95% ethanol for fixation and permeabilization, and dried onto slides. The sperm are then labeled either by indirect immunofluorescence using rabbit anti-human sperm antiserum or with fluoresceinated Pisum sativum agglutinin (PSA). Both probes intensely label the acrosomal region of acrosome-intact sperm. Electron microscopy revealed the major site of PSA binding to be the acrosomal contents. Acrosome-reacted sperm have diminished acrosomal labeling by both probes; sperm with nuclei labeled by Hoechst stain are considered nonviable, and are excluded from the assay. Both assays are rapid, give similar results, and detect an increase in acrosome reactions following exposure to the ionophore A23187.