Comparison of PAWP between separated spermatozoa based on density gradient centrifugation and Zeta potential

Density gradient centrifugation (DGC) and Zeta procedures are used for separation of normal sperm for treatment of infertility. Previous studies showed that Zeta method was more efficient in separation of normal sperm with intact chromatin compared to DGC. In this study, we aimed to compare postacrosomal sheath WW domain-binding protein (PAWP) protein as one of the factors involved to oocyte activation between two methods. Semen samples were collected from 23 men with normal sperm parameters (concentration, motility and morphology) according to World Health Organization criteria. Semen samples were washed with VitaSperm media and each sample were divided into three groups; control, DGC and Zeta. The percentage and intensity of PAWP were assessed by flow cytometry for each group. In addition, localization of PAWP in spermatozoa was detected by immunostaining procedure. Percentage of PAWP-positive spermatozoa was significantly higher in separated spermatozoa from DGC procedure in comparison with control and Zeta groups (P<0.001), while intensity of PAWP was significantly (P<0.001) higher in separated spermatozoa from Zeta method in comparison with control and DGC procedures. PAWP appear to be localized mainly in equatorial segment and post acrosome sheath of sperm head in all the groups. The Zeta method could separate low percentage of PAWP-positive spermatozoa compared to DGC method but such sperm appear higher intensity of PAWP than DGC. Therefore, separated spermatozoa by Zeta method possibly has better chance in activation of oocyte. However, further studies are needed to confirm this result.