Casey P J, Hillman R B, Robertson K R, Yudin A I, Liu I K, Drobnis E Z
Department of Reproduction, School of Veterinary Medicine, University of California, Davis 95616.
J Androl. 1993 Jul-Aug;14(4):289-97.
An acrosomal staining technique that can differentiate between living and dead sperm was developed for equine sperm. The fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) was used to identify the presence or absence of acrosomal contents, while the supravital nuclear dye Hoechst 33258 (H258) was used to assess viability. The accuracy of the FITC-PSA acrosomal stain was tested by comparing the percentage of sperm that had lost their acrosomal contents, detected by the staining method, with that detected by transmission electron microscopy (TEM). Following capacitation in vitro, the acrosomal status of sperm induced to acrosome react with A23187 and of control sperm were very similar with the staining technique and TEM, confirming the accuracy of the FITC-PSA acrosomal stain. We investigated the relationship between viability as measured by exclusion of H258 and motility as measured by three methods: one subjective and two objective. Although there was a good correlation between viability and motility as measured by all three methods (r = 0.88, 0.85, 0.75), there was always a proportion of viable sperm that were nonmotile. The physiology of the viable, nonmotile sperm was further investigated by comparing for individual sperm the viability as measured by exclusion of H258 with the mitochondrial function as measured by rhodamine 123. A good correlation (r = 0.99) was found to exist between viability and mitochondrial function, indicating that viable, nonmotile sperm possess functional mitochondria and confirming the ability of supravital staining to distinguish between living and dead sperm. We determined that 29-81% of the sperm in semen that had lost their acrosomal contents were in fact dead. Thus, this acrosomal staining technique can provide more relevant endpoints for future investigations of capacitation, the acrosome reaction, and sperm handling techniques in the horse.
我们开发了一种能区分马精子死活的顶体染色技术。用荧光素标记的凝集素豌豆凝集素（FITC-PSA）来识别顶体内容物的有无，同时用超活细胞核染料Hoechst 33258（H258）来评估活力。通过比较染色法检测到的顶体内容物缺失的精子百分比与透射电子显微镜（TEM）检测到的百分比，来测试FITC-PSA顶体染色的准确性。体外获能后，用A23187诱导顶体反应的精子和对照精子的顶体状态，在染色技术和TEM检测下非常相似，证实了FITC-PSA顶体染色的准确性。我们研究了通过H258排除法测定的活力与通过三种方法（一种主观方法和两种客观方法）测定的运动性之间的关系。尽管通过所有三种方法测定的活力和运动性之间都有良好的相关性（r = 0.88、0.85、0.75），但总有一部分有活力的精子是不运动的。通过比较单个精子通过H258排除法测定的活力与通过罗丹明123测定的线粒体功能，进一步研究了有活力但不运动精子的生理学。发现活力与线粒体功能之间存在良好的相关性（r = 0.99），表明有活力但不运动的精子具有功能性线粒体，并证实了超活染色区分死活精子的能力。我们确定，精液中顶体内容物缺失的精子中有29 - 81%实际上是死的。因此，这种顶体染色技术可为未来马的获能、顶体反应和精子处理技术的研究提供更相关的终点指标。
