Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage
Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage
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DOI：
10.1093/molehr/5.2.109
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发表时间：
1999-02-01
影响因子：
4
通讯作者：
Schaller, J
中科院分区：
文献类型：
--
作者：
Glander, HJ;Schaller, J
当细胞膜受到干扰时，磷脂酰丝氨酸（PS）从细胞膜的内叶向外叶转移。这是细胞凋亡的最早迹象之一，可以通过膜联蛋白v的钙依赖性结合来监测。因此，膜联蛋白v结合结合流式细胞术用于评估不同冷冻方案后精子质膜的完整性：即10% （v/v）甘油；精子维持培养基（MM）；冷冻培养基TEST蛋黄缓冲液（TYB）；或无保护的低温储存（低温休克）。使用膜联蛋白V和碘化丙啶（PI）两种荧光染料的组合，可以识别出三组精子：(i)有活力的精子（膜联蛋白V阴性和PI阴性）；（ii）死精子（膜联蛋白v阳性和pi阳性）；（iii）质膜受损但完整的细胞（膜联蛋白v阳性和pi阴性）。经冷冻休克处理的精子存活膜联蛋白v阴性精子比例（15.0 +/- 1.2%）与经10% （v/v）甘油（19.9 +/- 1.6%）和MM（22.2 1.8%）冷冻保存的TYB冷冻精子比例（26.6 +/- 2.2%）显著增加（P < 0.05），并与活动精子比例（甘油17.6 +/- 3.4%、MM 19.6 +/- 3.7%和TYB 22.6 +/- 3.9%）相关（P 0.0001）。在精子中，即使它们不与PI结合，也有12-22%的膜联蛋白v阳性，表明冷冻前和冷冻后的活力。活膜联蛋白v阳性精子百分比与不同精子运动参数呈显著相关（速度呈直线，r = 0.601, P = 0.018；线性运动精子百分比：r = 0.549, P = 0.034）。因此，我们得出结论，膜联蛋白v结合在检测膜功能恶化方面比PI染色更敏感，并且除了死亡或垂死的细胞外，相当大比例的精子可能具有功能失调的质膜。在测试的冷冻保存方案中，TYB产生的精子存活率最高。因此，我们提倡使用膜联蛋白v结合试验来评估精子的质量和完整性。
When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0 +/- 1.2%) to spermatozoa cryopreserved by TYB (26.6 +/- 2.2%) via cryopreservation by 10% (v/v) glycerol (19.9 +/- 1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6 +/- 3.4% by glycerol; 19.6 +/- 3.7% by MM and 22.6 +/- 3.9% by TYB; P 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa.