In addition to the proteins enriched in membrane rafts, DRMs are enriched in gangliosides. Recently, fluorescently labeled cholera toxin has been used to address the presence of GM1 in sperm from different mammalian species [19, 21, 23, [30] [31] [32] [33] . Although GM1 is the best studied in sperm, other gangliosides are known to be enriched in membrane rafts [34] , and differential ganglioside distribution among these membrane microdomains has been reported [35] [36] [37] . In particular, GM3 appears to be the major ganglioside in the male reproductive system [38] , and several studies [39] [40] [41] demonstrate its abundance in bovine, ovine, and human sperm. However, localization of GM3 on sperm has not yet been analyzed, to our knowledge. To investigate the location of GM3 in mouse sperm, a specific anti-GM3 antibody was tested by immunofluorescence. This monoclonal antibody has been validated by Kotani et al. [42, 43] and was used in several studies [35, [44] [45] [46] [47] [48] . Anti-GM3 fluorescent signal was found to be restricted to the equatorial segment (Fig. 4) , whereas PNA signal exclusively stained the anterior acrosomal region. 4 . Detection of ganglioside GM3 in mouse sperm. Mouse sperm fixed and permeabilized as already described were incubated overnight with a monoclonal anti-GM3 antibody, followed by Alexa Fluor 555-conjugated secondary antibody (GM3). Cells were also stained with Alexa Fluor 488-conjugated PNA. No primary antibody controls were used (lower panels). Experiments were repeated at least three times; representative sperm are shown. DIC, differential interference contrast. Original magnification 360.