Characterization of two heterozygous mutations of the oocyte activation factor phospholipase C zeta (PLCζ) from an infertile man by use of minisequencing of individual sperm and expression in somatic cells  Junaid Kashir, Ph.D., Michalis Konstantinidis, B.Sc., Celine Jones, Bjorn Heindryckx, Ph.D., Petra De Sutter, M.D., Ph.D., John Parrington, Ph.D., Dagan Wells, Ph.D., Kevin Coward, Ph.D.  Fertility and Sterility  Volume 98, Issue 2, Pages 423-431 (August 2012) DOI: 10.1016/j.fertnstert.2012.05.002 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative minisequencing peaks of single sperm from the patient exhibiting phospholipase C zeta (PLCζ) mutation for (A) PLCζH233L or (B) PLCζH398P. Single sperm exhibited either an adenine peak (Ai) or a thymine peak (Aii) upon screening for PLCζH233L, or an adenine peak (Bi) or a cytosine peak (Bii) upon screening for PLCζH398P. Y-axis: relative fluorescence units; X-axis: amplicon base number. Fertility and Sterility 2012 98, 423-431DOI: (10.1016/j.fertnstert.2012.05.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Representative confocal microscopy images taken across 48- and 56-hour time points for HEK293T cells transfected with mCherry-PLCζ constructs. Images were captured at a magnification of ×63, and white scale bars indicate 10 μm. (B) Mean mCherry fluorescence intensity per cell for HEK293T cells transfected with mCherry PLCζWT, PLCζH398P, PLCζH233L, and PLCζH233L+H398P at 48- and 56-hour time points. Data are shown as mean ± standard error of the mean. Asterisks (*,**,***) and hash marks (#,###) on the histograms indicate a statistically significant difference (P<.05). Differences between fluorescence levels not annotated represent a statistically insignificant (P≤.05) difference. Fertility and Sterility 2012 98, 423-431DOI: (10.1016/j.fertnstert.2012.05.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Schematics showing a possible model of spermatogenesis in (A) the patient's father and (B) the patient, indicating how both PLCζH233L and PLCζH398P DNA and protein may be divided among the sperm population, assuming that human phospholipase C zeta (PLCζ) is transcribed throughout the meiotic phases of spermatogenesis, resulting in sperm with mixed populations of PLCζ protein, where normal PLCζ protein may override the deleterious effects of mutated PLCζ, thereby rescuing the oocyte-activating ability of the affected sperm. Fertility and Sterility 2012 98, 423-431DOI: (10.1016/j.fertnstert.2012.05.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions