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首页> 外文期刊>Molecular reproduction and development >Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by Dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca2+ch
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Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by Dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca2+ch

机译：透明带溶解诱导小鼠精子顶体反应的动力学：荧光定量法测定达波西尔（2-氨乙基）磺酰胺和细胞内Ca2 + ch监测顶体腔与培养基之间pH梯度损失

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The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the 3-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, DapoxylTM (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13±0.10 min (n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca2+ concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85±0.09 min (n = 6) to a steady state level higher than the initial level, indicating this Ca2+ transient as the precursor reaction to onset of the zona-induced acrosome reaction.
机译：透明质酸诱导的小鼠精子顶体反应的开始的特征是，由于顶体和质膜之间的孔形成，酸性顶体管腔和悬浮介质之间的顶体完整精子中存在的pH梯度下降。在较早的工作中，表明这种pH梯度的损失发生在单个精子上，该单个精子与结构完整的透明小带结合在一起的时间为2.1分钟;在与完整的无性系相关的精子群体中确定的这种损失的延长动力学是由于180分钟的可变滞后时间范围。我们假设该滞后时间范围是由于结构完整的透明带的3维结构所施加的空间限制，因此应在溶解的带状疱疹中消除此限制。荧光探针DapoxylTM（2-氨基乙基）磺酰胺（DAES）可以在精子细胞群中验证这一假设。它是一种弱碱，在水溶液中不发荧光，但由于pH梯度增加，荧光增强，因此在酸性顶体隔室中积聚。 pH梯度的损失会导致荧光减弱。在能力增强的顶体完整精子中，对溶解的透明带蛋白的响应，DAES荧光损失的半衰期为2.13±0.10分钟（n = 9）。单细胞和细胞群体动力学之间的一致性验证了结构完整的透明带中空间约束的假说。如fluo-3所监测，响应于增溶的透明带的细胞内Ca2 +浓度的变化是迅速增加，然后是下降，中间时间为0.85±0.09分钟（n = 6）至稳态水平高于初始水平，表明此Ca2 +瞬变是Zona诱导的顶体反应发作的前体反应。

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