Nuffield Department of Women's and Reproductive Health, University of Oxford, Oxford OX3 9DU, UK.
The Women's Centre, Oxford University Hospitals NHS Foundation Trust, Oxford OX3 9DU, UK.
Asian J Androl. 2022 Jul-Aug;24(4):345-352. doi: 10.4103/aja202168.
Phospholipase C zeta (PLCζ) is a sperm-specific protein that triggers oocyte activation. The analysis of PLCζ expression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency. Our laboratory has previously optimized a standard "in-house" assay to determine PLCζ expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLCζ in human sperm. This study aimed to compare our established assay and AUM (involving HCl, acidic Tyrode's solution [AT], and heat). The mean relative fluorescence (RF) intensity of PLCζ in frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups (in-house [mean ± standard error of mean]: 18.87 ± 2.39 arbitrary units [a.u.] vs non-AUM: 11.44 ± 1.61 a.u., AT-AUM: 12.38 ± 1.89 a.u., and HCl-AUM: 12.51 ± 2.16 a.u., P < 0.05, one-way analysis of variance). The mean RF intensity of PLCζ in AT- and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group. However, the in-house method resulted in the highest RF intensity (12.11 ± 1.36 a.u., P < 0.01). Furthermore, specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM. In conclusion, our in-house method showed superior visualization and reliability than the AUM, thus supporting the continued use of our in-house assay for clinical research screening.
PLCζ 是一种精子特异性蛋白，可触发卵母细胞激活。分析人精子中 PLCζ 的表达可以作为卵母细胞激活缺陷的诊断标志物。我们实验室之前已经优化了一种标准的“内部”检测方法，用于检测人精子中 PLCζ 的表达。然而，有一项研究表明，抗原暴露方法（AUM）在人精子中可视化 PLCζ 会更有效。本研究旨在比较我们已建立的检测方法和 AUM（涉及 HCl、酸性 Tyrode 溶液[AT]和热）。十四位健康供体的冷冻解冻精子用内部方法染色后 PLCζ 的平均相对荧光（RF）强度明显高于其他三个 AUM 组（内部[平均值±标准误差均值]：18.87±2.39 任意单位[a.u.]与非 AUM 组：11.44±1.61 a.u.，AT-AUM 组：12.38±1.89 a.u.和 HCl-AUM 组：12.51±2.16 a.u.，P<0.05，单因素方差分析）。十二位不育男性的 AT 和 HCl 处理精子的 PLCζ 平均 RF 强度与非 AUM 组无显著差异。然而，内部方法导致的 RF 强度最高（12.11±1.36 a.u.，P<0.01）。此外，抗体-抗原结合的特异性测试表明，内部方法显示出比 AUM 处理的精子更强的特异性结合。总之，与 AUM 相比，我们的内部方法在可视化和可靠性方面表现出优势，因此支持继续将我们的内部检测用于临床研究筛选。
