Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology  Mahmoud Aarabi, M.D. Ph.D., Hanna Balakier, Ph.D., Siamak Bashar, Ph.D., Sergey I. Moskovtsev, M.D., Ph.D., Peter Sutovsky, Ph.D., Clifford L. Librach, M.D., Richard Oko, Ph.D.  Fertility and Sterility  Volume 102, Issue 2, Pages 440-447 (August 2014) DOI: 10.1016/j.fertnstert.2014.05.003 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Human PAWP is expressed during spermatid elongation and assembled in the PAS-PT. (A) Representative immunoblotting of sperm samples from normal fertile donors with antibodies against human PAWP (left) and human PLCζ (right). Treatment of whole sperm (WS) with nonionic detergent TX released PLCζ into the detergent-soluble compartment, while PAWP remained in the detergent-resistant pellet (Pel). (B) PAWP immunofluorescence is localized to the PAS-PT of mature spermatozoa from a fertile donor. In contrast, very little immunofluorescence is detected in the sperm from a patient with globozoospermia, suggesting abnormal PAWP expression or assembly in such patients. Bars = 10 μm. (C) Light micrographs of human seminiferous tubules. Immunoperoxidase stained with anti-human PAWP antibody, demonstrating PAWP's expression during spermiogenesis. The highest immunoreactivity is detected in elongating spermatids (ES) where PAS-PT is formed, while very little PAWP protein is detected in round spermatids (RS). Bar = 40 μm. Fertility and Sterility 2014 102, 440-447DOI: (10.1016/j.fertnstert.2014.05.003) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Representative demonstrations of PAWP expression in the spermatozoa from men with high (>70%; a) and low (<20%; b) ICSI fertilization rates. (A) immunofluorescence signal of PAWP was detected in most of the sperm from patients with high ICSI fertilization rates in contrast to little or no signal in the sperm of the patient with low ICSI fertilization rates. Bars = 10 μm. (B) Overlay histograms of gated sperm populations from flow-cytometric analysis of patient samples (red) and corresponding controls incubated with the preimmune serum (gray) reveal high levels (>70%) versus very low levels (<10%) of PAWP in patients a and b, respectively. (C) Corresponding Western blot of sperm samples a and b, immunoblotted with anti-human PAWP antibody, demonstrating relative levels of PAWP correlating to flow cytometry results. Fertility and Sterility 2014 102, 440-447DOI: (10.1016/j.fertnstert.2014.05.003) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 ICSI fertilization rates and human embryonic development are correlated with flow-cytometric PAWP levels. Scatter plots show a statistically significant correlation between PAWP-induced fluorescence and ICSI fertilization rates (A; r = 0.73, P<.0005), as well as embryonic arrest rates (B; r = −.31, P=.001). Fertility and Sterility 2014 102, 440-447DOI: (10.1016/j.fertnstert.2014.05.003) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions