HYPEROSMOTIC TOLERANCE OF HUMAN SPERMATOZOA - SEPARATE EFFECTS OF GLYCEROL, SODIUM-CHLORIDE, AND SUCROSE ON SPERMOLYSIS
HYPEROSMOTIC TOLERANCE OF HUMAN SPERMATOZOA - SEPARATE EFFECTS OF GLYCEROL, SODIUM-CHLORIDE, AND SUCROSE ON SPERMOLYSIS
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DOI：
10.1095/biolreprod49.1.112
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发表时间：
1993-07-01
影响因子：
3.6
通讯作者：
CRITSER, JK
中科院分区：
文献类型：
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作者：
GAO, DY;ASHWORTH, E;CRITSER, JK
细胞在冷冻过程中所经历的高渗应激以及在升温过程中高渗应激的释放都与冷冻损伤有关。为了确定最佳的冷却或升温速率并防止对人类精子的渗透损伤，需要关于细胞的渗透耐受性的信息作为1）时间、2）温度、3）溶质类型和4）溶质浓度的函数。将人类精子样本分成三等份。将等分试样分别在0、8和22 ℃下平衡。通过将渗透性冷冻保护剂（甘油）或非渗透性溶质（蔗糖，非离子型;或NaCl，离子型）添加到等渗Mann’s Ringer溶液中，制备不同的高渗溶液。将制备的溶液的等分试样分别在0、8和22 ℃下平衡。在相应的温度下，将小体积（2.5穆尔）的每种精子等分试样与50穆尔的每种高渗溶液快速混合。在5秒至5分钟的时间范围后，通过在相应温度下与500穆尔曼氏林格氏溶液混合，将10穆尔每种高渗细胞悬浮液突然返回到等渗环境。通过双重染色（二乙酸羧荧光素和碘化丙啶）技术和流式细胞术测量暴露于高渗应激后和返回等渗条件后细胞的质膜完整性。用冷冻精子扫描电镜观察了处理后精子细胞的形态，结果表明，人精子在冷冻后出现了明显的溶解/损伤，当暴露于NaCl或蔗糖的高渗溶液后恢复到等渗条件时，膜完整性丧失。高渗浓度越高，细胞损伤越严重。当渗透压为2000 mOsm时，大多数细胞（> 50%）丧失膜完整性。相反，如果精子没有回到等渗条件下，高渗溶液中的大多数精子似乎保持膜的完整性。对于一个给定的较高的高渗（> 1000 mOsm），posthypypertonic精子随着温度的降低而减少。细胞存活也受到细胞暴露于高渗环境的时间的影响，然后细胞返回到等渗条件。时间越短，细胞存活率越高。当暴露于相对于电解质等渗的高渗甘油溶液时，如果甘油的渗透压摩尔浓度< 3000 mOsm，则很少有细胞丧失其膜完整性。对于固定的高渗透压（> 3000 mOsm），温度越低，精子百分比越高。在本研究中的最高甘油浓度下，即，4694 mOsm时，在0 ℃、8 ℃和22 ℃下，精子百分比分别为17%、10%和2%。从细胞中去除甘油所引起的精子变性取决于使细胞恢复到等渗条件的方法。一步恢复到等渗条件导致严重的精液不足，而多步（九步）程序显着减少精液不足。扫描电子显微镜照片显示了不同渗透条件下精子的不同形态。精子的异常进行posthypertonic治疗显示得特别清楚。
Hyperosmotic stress, which cells experience during the freezing process, and its release during the warming process are both related to cryoinjury. To define optimal cooling or warming rates and prevent osmotic injury to human sperm, information is required regarding the osmotic tolerance of the cells as a function of 1) time, 2) temperature, 3 ) type of solute, and 4) solute concentration. Human sperm samples were divided into three aliquots. The aliquots were equilibrated at 0, 8, and 22-degrees-C, respectively. Different hyperosmotic solutions were prepared by addition of either a permeating cryoprotective agent (glycerol) or nonpermeating solutes (sucrose, non-ionic; or NaCl, ionic) to isotonic Mann's Ringer solution. Aliquots of the prepared solutions were equilibrated at 0, 8, and 22-degrees-C, respectively. A small volume (2.5 mul) of each sperm aliquot was quickly mixed with 50 mul of each hyperosmotic solution at the corresponding temperature. After times ranging from 5 s to 5 min, 10 mul of each hyperosmotic cell suspension was abruptly returned to an isosmotic environment by mixing with 500 mul of Mann's Ringer solution at the corresponding temperature. The plasma membrane integrity of cells after exposure to hyperosmotic stress and after return to isosmotic conditions was measured by a dual staining (carboxyfluoroscein diacetate and propidium iodide) technique and flow cytometry. The morphology of the treated cells was observed by scanning electron microscopy of freeze-substituted sperm.The results indicate that human spermatozoa exhibited a significant posthypertonic lysis/injury, i.e., loss of membrane integrity, when returned to isosmotic conditions after exposure to hyperosmotic solutions of NaCl or sucrose. The higher die hyperosmolality, the more serious the cell injury. The majority of the cells (> 50% ) lost membrane integrity when die osmolality was a 2000 mOsm. In contrast, if the sperm were not returned to isosmotic conditions, the majority of the sperm in the hyperosmotic solutions appeared to maintain membrane integrity. For a given higher hyperosmolality (> 1000 mOsm), posthypertonic spermolysis was reduced with a decrease of temperature. Cell survival was also affected by time of cell exposure to hyperosmotic environments before cells were returned to the isotonic condition. The shorter the time, the higher the cell survival. When exposed to hyperosmotic glycerol solutions that were isotonic with respect to electrolytes, few cells lost their membrane integrity if the osmolality of glycerol was < 3000 mOsm. For a fixed high osmolality (> 3000 mOsm), the lower the temperature, the higher the percentage spermolysis. At the highest glycerol concentration in this study, i.e., 4694 mOsm, the percentage spermolysis was 17%, 10%, and 2% at 0-degrees-C, 8-degrees-C, and 22-degrees-C, respectively. Spermolysis caused by the removal of glycerol from the cells depended on the means by which the cells were returned to isotonic conditions. A one-step return to isotonic conditions resulted in serious spermolysis, while a multi-step (nine-step) procedure significantly reduced the spermolysis. The scanning electron micrographs showed the distinct morphology of the spermatozoa experiencing the different osmotic conditions. The abnormality of spermatozoa that underwent posthypertonic treatment was demonstrated especially clearly.