8 FLOW CYTOMETRIC MONITORING OF CHOLERA TOXIN B SUBUNIT BINDING TO BOVINE SPERMATOZOA

In order to become able to fertilize, mammalian spermatozoa must undergo a series of biochemical modifications. This process called capacitation involves several changes of the content and the ultrastucture of the plasma membrane. Among these changes, loss of cholesterol from the plasma membrane is required. Lipid rafts are detergent-insoluble plasma membrane domains rich in cholesterol and sphingolipids. Some proteins are confined to lipid rafts while others are excluded. It has been hypothesized in the past that the loss of cholesterol could destabilize and relocate lipid rafts and would thus affect protein interactions in the plasma membrane, thereby leading to downstream events involved in the capacitation process. Thus, quantification of lipid rafts within the membrane of spermatozoa would become useful to monitor sperm functions and maturation level. The present study aimed to quantify lipid rafts in bovine spermatozoa using the Vibrant Lipid Raft detection kit from Molecular Probes (Invitrogen Canada, Inc., Burlingame, Ontario, Canada) and flow cytometry. The Vibrant kit uses the cholera toxin B subunit (CT-B) and claims to detect ganglioside Gm1 that sublocalizes within lipid rafts. Briefly, freshly ejaculated and frozen/thawed spermatozoa were washed once by centrifugation at 250g for five min in sp-Talp and were then re-suspended in sp-Talp containing 1 �g mL–1 CT-B. Then, cells were incubated at 4�C for 10 min, washed in chilled sp-Talp, incubated for 15 min in the presence of an anti-CT-B antibody coupled to the Alexa Fluor� 488 dye (Molecular Probes), and washed again to remove excess antibody. Spermatozoa were then analyzed with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Two populations showing different fluorescence levels were observed in all samples. Greater proportions of spermatozoa displayed the high fluorescence pattern in cryopreserved samples (37.9%) when compared to freshly ejaculated spermatozoa (8.2%) (P < 0.01). Also, when compared to freshly ejaculated spermatozoa, increased proportions of high fluorescence was detected following a 6-h incubation in sp-Talp containing bicarbonate and BSA. These results suggest that capacitation and cryopreservation both promote exposure of CT-B binding molecules in bovine spermatozoa. Microscopic observation of labeled cryopreserved spermatozoa did not yield the expected raft labeling patterns, but rather 5 different patterns of labeling. In the past, some of these patterns were recognized to be associated with capacitation and acrosome reaction. At this point, more work is needed to confirm which of the fluorescent patterns observed in microscopy corresponds to the enhanced fluorescence sperm population observed by flow cytometry and to directly associate this enhanced fluorescence to capacitation or the acrosome reaction. In conclusion, it appears that the Vibrant kit from Molecular Probes cannot be used to quantify lipid rafts by flow cytometry. Nevertheless, it might be an interesting tool to use in flow cytometry to monitor membrane changes associated with capacitation or cryo-damage.