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Volume 24, Issue 4 (July 2022) 24, 345–352; 10.4103/aja202168

Antigen unmasking does not improve the visualization of phospholipase C zeta in human spermatozoa

Xin Meng1, Celine Jones1, Pedro Melo2, Caroline Ross3, Ginny Mounce1, Tim Child1,3, Kevin Coward1

1 Nuffield Department of Women's and Reproductive Health, University of Oxford, Oxford OX3 9DU, UK
2 The Women's Centre, Oxford University Hospitals NHS Foundation Trust, Oxford OX3 9DU, UK
3 Oxford Fertility, Oxford Business Park North, Oxford OX4 2HW, UK

Correspondence: Dr. K Coward (kevin.coward@wrh.ox.ac.uk)

Date of Submission 27-Oct-2020 Date of Acceptance 01-Sep-2021 Date of Web Publication 03-Dec-2021


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 ORIGINAL ARTICLE, 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.

Keywords: antigen unmasking; immunofluorescence staining; male infertility; phospholipase C zeta

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