CENTRIFUGATION OF HUMAN SPERMATOZOA INDUCES SUBLETHAL DAMAGE - SEPARATION OF HUMAN SPERMATOZOA FROM SEMINAL PLASMA BY A DEXTRAN SWIM-UP PROCEDURE WITHOUT CENTRIFUGATION EXTENDS THEIR MOTILE LIFETIME
CENTRIFUGATION OF HUMAN SPERMATOZOA INDUCES SUBLETHAL DAMAGE - SEPARATION OF HUMAN SPERMATOZOA FROM SEMINAL PLASMA BY A DEXTRAN SWIM-UP PROCEDURE WITHOUT CENTRIFUGATION EXTENDS THEIR MOTILE LIFETIME
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DOI：
10.1093/oxfordjournals.humrep.a138198
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发表时间：
1993-07-01
影响因子：
6.1
通讯作者：
STOREY, BT
中科院分区：
文献类型：
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作者：
ALVAREZ, JG;LASSO, JL;STOREY, BT
虽然通过离心和再悬浮洗涤人类精子细胞是一种广泛使用的程序，但有迹象表明，这种程序本身可能对细胞有害。本研究的目的是调查这个问题。为此，开发了一种在不需要离心的情况下从精浆中干净分离活动人类精子的方法，使用改进的游动程序，将液化精液与等体积的30 mg/ml葡聚糖混合在培养基中，并将混合物覆盖在含有5 mg/ml牛血清白蛋白的培养基上，形成两层具有稳定界面的离散层。在一个给定的样品中，恢复后立即运动细胞的百分比始终保持在80%左右。通过在恢复后96小时的孵育期间运动细胞的丧失来评估细胞的损伤。比较右旋糖酐泳装获得的精子，离心后48 h有4个+/- 3%的运动细胞，而未处理的精子有52个+/- 12%的运动细胞。恢复后1 h，等价物无差异。精子在精浆中离心，在新鲜血浆中重悬，然后用葡聚糖游泳回收，也得到了类似的结果。在离心样品中迟发性运动丧失表明这种处理诱导了细胞的亚致死损伤。精子恢复的标准游动法和Percoll梯度法（这两种方法都涉及到离心步骤）的比较表明，样品活力的下降与右旋糖酐游动法所见的相似。我们得出的结论是，与处理方法无关，离心本身会引起人精子的亚致死损伤，并且表明避免离心的人精子恢复方法可能会部分减轻这些细胞在冷冻保存期间造成的损伤。
While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently >80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.