Cheng F P, Fazeli A, Voorhout W F, Marks A, Bevers M M, Colenbrander B
Department of Herd Health & Reproduction, Veterinary Faculty, Utrecht University, The Netherlands.
J Androl. 1996 Nov-Dec;17(6):674-82.
Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.
花生凝集素（PNA）用于评估马属动物配子相互作用过程中的精子顶体状态和顶体反应。通过透射电子显微镜证实，PNA仅与种马精子的顶体外膜结合。异硫氰酸荧光素-PNA（FITC-PNA）标记用于监测精子在24小时的延长孵育期以及在存在5微摩尔钙离子载体A23187的情况下2小时孵育期间的顶体变化。此外，在SP-TALP培养基中预孵育4小时后，将精子样本与匹配的半透明带孵育1分钟（起始结合），然后在无精子培养基中将精子-半透明带复合物孵育60分钟（1小时结合），以评估结合精子的顶体状态。为了评估顶体，将精子和洗涤后的精子-半透明带复合物空气干燥到载玻片上，固定，用乙醇通透处理，用FITC-PNA染色，并用DNA染料乙锭同二聚体复染。与透明带结合和未结合的精子都显示出相似的染色模式。顶体完整的精子在顶体帽上显示强烈的绿色荧光，而发生反应的精子则显示出荧光的斑驳破坏图像。完成顶体反应的精子细胞主要在赤道段染色或根本不染色。在延长孵育期间和钙离子载体处理期间，顶体发生修饰（反应）和顶体完全分解（已反应）的精子比例显著增加。在精子与透明带接触的60分钟内观察到顶体反应的显著诱导（P < 0.001）。总之，本研究证明了对种马精子在透明带表面的顶体状态和顶体反应发生率进行快速可靠评估的方法。
