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Shana Robson

Fellow – Shana Robson

Mentor – Dickson Varner

Project – Evaluation of techniques to differentiate acrosomal damage from acrosome reaction in stallion spermatozoa

Objective – The aim of this study is to validate two assays that might more critically assess acrosomal status.

Background – Prior to fertilization of the oocyte, sperm must undergo the acrosome reaction. This reaction brings about vesiculation between the outer acrosomal membrane and the overlying plasma membrane; thereby creating pores that allow release of enzymes from the acrosomal matrix that are necessary for penetration of the vestments of an oocyte. The acrosome reaction is preceded by a process of cholesterol efflux from the sperm head, allowing the rearrangement of fusogenic lipids and lipid rafts necessary for the acrosome reaction and binding of the sperm to the zona pellucida. The current fluorescent assay for detection of the acrosome reaction utilizes a lectin which binds to carbohydrates within the acrosomal matrix. This assay is unable to differentiate between the physiologic acrosome reaction and acrosomal damage caused by other factors, such as cryopreservation. We hypothesize that an efflux of cholesterol in acrosome reacted sperm will result in decreased staining as compared to sperm with acrosomal damage. Similarly, we hypothesize that lipid rafts and, therefore, Vybrant staining, will move from the base to the apical surface of the sperm head in capacitated sperm, but not acrosome damaged sperm. Validation of these two assays would add a new dimension to examination of semen quality in breeding stallions.

Procedure – Two stains are to be examined: filipin, which binds to cholesterol; and Vybrant Alexa Fluor 488, which binds to lipid rafts. Stained sperm samples will be analyzed using the ImageStream Imaging Flow Cytometer (Amnis Corporation, Seattle, WA), which combines flow cytometry and fluorescence microscopy in a single platform.

Results – there was a decrease in staining in the A23187 treated samples than in the untreated samples for both the DMSO and EtOH diluted filipin. Futher analysis is required to evaluate results, compensate the analysis to the stain, and begin further testing with the Vybrant.

Conclusions – The preliminary results provide a basis for further testing with these two stains. A decrease in filipin staining was seen in spermatozoa treated with A23187 as compared to samples not treated. This indicates that a cholesterol efflux occurs during the acrosome reaction and supports Gadella’s hypothesis that the cholesterol efflux is indeed related to the acrosome reaction, not just capacitation.