Ambrose J D, Rajamahendran R, Lee C Y
Department of Animal Science, University of British Columbia, Vancouver, Canada.
J Androl. 1996 Sep-Oct;17(5):579-86.
Anti-bull sperm monoclonal antibodies (mAbs), generated against intra-acrosomal and surface antigens, were evaluated for their functional significance. In experiment I, the influence of mAbs on the bovine sperm-oocyte interaction in vitro was tested on a total of 493 oocytes in either three or four replicate trials. Although the number of sperm bound per zona increased significantly over untreated control samples (23.6 +/- 5.6 vs. 10.0 + 2.4, mean +/- standard error [SE]; P < 0.001) in the presence of one surface-reacting mAb, other mAbs had no effect. Experiment II was designed to determine if the mAbs would detect capacitation-related changes of bull sperm in vitro. Bull sperm were incubated in capacitation medium (supplemented Tyrode's medium [TALP] plus 10 micrograms/ml heparin) for up to 4.5 hours. At 0 and at 4 hours, mAbs in hybridoma culture supernatant were incubated for 30 minutes with sperm, labeled with fluorescein isothiocyanate (FITC)-conjugated secondary antibody, and processed for indirect immunofluorescence assay. Four mAbs specific to intra-acrosomal antigens exhibited a time-dependent increase (P < 0.05) in binding to bull sperm incubated under capacitation conditions. In contrast, the binding of the mAbs specific to surface antigens significantly decreased (P < 0.05) after 4 hours in the presence of heparin. Sperm viability did not change during the 4-hour period. In experiment III, mAbs specific to intra-acrosomal antigens were evaluated to assess bull sperm acrosomal status following lysophosphatidylcholine-induced acrosome reaction. A significant decrease (P < 0.01) in mAb binding following the induced acrosome reaction was observed with all the mAbs; it was highly correlated (r > or = 0.85; P < 0.01) with Pisum sativum agglutinin binding and Giemsa staining. The results suggest that some of the mAbs are potential biological markers for bull sperm surface changes associated with capacitation and the acrosome reaction in vitro.
针对顶体内和表面抗原产生的抗公牛精子单克隆抗体（mAb），对其功能意义进行了评估。在实验I中，在三到四次重复试验中，对总共493个卵母细胞测试了mAb对体外牛精子 - 卵母细胞相互作用的影响。虽然在存在一种表面反应性mAb的情况下，每个透明带结合的精子数量比未处理的对照样品显著增加（23.6±5.6对10.0 + 2.4，平均值±标准误差[SE]；P <0.001），但其他mAb没有效果。实验II旨在确定mAb是否能检测体外公牛精子与获能相关的变化。将公牛精子在获能培养基（添加的Tyrode培养基[TALP]加10微克/毫升肝素）中孵育长达4.5小时。在0小时和4小时时，将杂交瘤培养上清液中的mAb与精子孵育30分钟，用异硫氰酸荧光素（FITC）偶联的二抗标记，并进行间接免疫荧光测定。四种针对顶体内抗原的mAb在获能条件下孵育的公牛精子上的结合呈现时间依赖性增加（P <0.05）。相反，在肝素存在下4小时后，针对表面抗原的mAb的结合显著降低（P <0.05）。精子活力在4小时期间没有变化。在实验III中，评估了针对顶体内抗原的mAb，以评估溶血磷脂酰胆碱诱导的顶体反应后公牛精子的顶体状态。在诱导的顶体反应后，观察到所有mAb的mAb结合均显著降低（P <0.01）；它与豌豆凝集素结合和吉姆萨染色高度相关（r≥0.85；P <0.01）。结果表明，一些mAb是体外与获能和顶体反应相关的公牛精子表面变化的潜在生物学标志物。
