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

Phospholipase C zeta (PLC zeta) is a sperm-specific protein that triggers oocyte activation. The analysis of PLC zeta 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 PLC zeta expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLC zeta 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 PLC zeta 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 PLC zeta 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.