流式细胞术分析面对凝集的种马精子膜完整性：“僵尸精子”的困境。
Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the "zombie sperm" dilemma.
机构信息
College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Department of Clinical Studies-New Bolton Center, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA, 19348, USA.
出版信息
J Assist Reprod Genet. 2021 Sep;38(9):2465-2480. doi: 10.1007/s10815-021-02134-z. Epub 2021 May 15.
PURPOSE
To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect.
METHODS
Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions.
RESULTS
Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results ("zombie sperm"). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0.
CONCLUSION
Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.
目的
定义在获能条件下孵育时精子凝集对通过流式细胞术评估膜准确性的影响，并开发减轻这种影响的方法。
方法
使用 CASA 测量精子活力。精子用 PI-PSA 或一种新方法 LD-PSA 染色，使用可固定活/死染色和细胞解离处理，然后进行流式细胞分析。使用 LD-PSA，在 10μm A23187 处理 10 分钟后，确定马精子的顶体反应和质膜状态，然后在获能条件下孵育 0、1 或 2 小时。
结果
使用 PI-PSA，测量的膜完整性（活精子）明显低于总活力（TMOT），表明存在虚假结果（“僵尸精子”）。精子聚集体主要是运动精子。A23187 处理后丧失活力与解聚和增加的 MI 有关。用 LD-PSA 解聚后，MI 上升，MI 然后与 TMOT 对应。在 A23187 处理后孵育的马精子中，随着活顶体反应精子的百分比增加，TMOT 接近 0。
结论
流式细胞术仅评估单个精子；因此，活精子的凝集改变了记录的膜完整性。随着有活力的精子变得不动，它们就会个体化；因此，降低活力的因素，如 A23187，会导致测量的 MI 增加。在评估前进行解聚可以更准确地确定精子膜状态；在这种情况下，我们记录了与马 IVF 中 A23187 处理的重复性差相关的运动和活顶体反应的马精子之间的不匹配，这些发现对未来的精子获能研究具有深远的价值。
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