Phospholipase C-Zeta Reveals the Underlying Pathological Influence after Density Gradient Centrifugation, Microfluidic Sorting, and Cryopreservation of Human Sperm

Objective:Sperm preparation techniques and cryopreservation are widely used in assisted reproductive techniques(ART).How to improve the quality of sperm management is a matter of great concern.Phospholipase C-zeta(PLCζ)is considered a sperm-specific agent that activates oocyte activation and thus playing a crucial role in male fertility.However,the potential mechanisms by which semen processing and cryopreservation on PLCζcontribute to keyhole have not been addressed.Methods:In this study,semen samples were taken from have not been addressed 10 normozoospermic men.Each semen sample was assigned to the following groups:density gradient centrifugation(DGC)as control,microfluidic sorting,and cryopreservation.Sperm parameters of molity,viability,membrane integrity,and intracellular ROS were evaluated during sperm preparation and cryopreservation.The expression of PLCζin human sperm was determined by immunofluorescence and western blotting.Results:The results showed that molity,viability,and membrane integrity decreased in cryopreservation group.Intracellular ROS were also significantly increased compared to the the control group.There was no significant difference between DGC and microfluidic sorting group.Our investigation revealed that total levels of PLCζwere comparable between DGC and microfluidic sorting,but there were significantly reduced levels of PLCζafter cryopreservation as quantified by both immunofluorescenceand immunoblotting.PLCζimmunofluorescence in sperm revealed different PLCζlocalization patterns around the acrosomal(Ac),equatorial(Eq),post-acrosomal(PA)areas of sperm heads,and their combination.The predominant patterns of PLCζlocalization in DGC were similar to that of microfluidic sorting,with strong,with staining.In contrast,PLCζstaining in freeze-thawed sperm was considerably weaker fluorescence intensity.Conclusion:This study clarified the mechanism of sperm preparation and cryopreservation underlying effect on sperm characteristic,accompanied with PLCζexpresion.We demonstrated that microfluidic sorting provides a highly efficient preparation method for clinical selection of PLCζ-expressing sperm comparable to DGC gene expression.It is suggested that the cryopreservation of sperm has a significant detrimental effect on PLCζ.