Fig. 2

Epididymosomes transfer biotinylated proteins to the head of caput epididymal spermatozoa. Mouse caput spermatozoa were co-cultured with biotin-labeled epididymosomes in vitro for 1 h prior to being fixed and labeled with streptavidin conjugated to Alexa Fluor 488 to detect the spatial profile of transferred biotinylated proteins. a, d Prominent post-acrosomal domain staining was observed for both biotin reagents, while e whole head and mid-piece staining was exclusively observed in a small portion of cells incubated with epididymosomes biotinylated with the membrane permeant reagent. Representative controls are included in which spermatozoa were either directly labeled with b membrane impermeant biotin or f the membrane permeant biotin, both of which yielded a distinct profile of biotinylation across the entire spermatozoon. c, g Spermatozoa were also incubated with non-biotinylated epididymosomes as a negative control to confirm the absence of endogenous biotin signal under the same imaging conditions. All experiments were repeated at least three times and representative images are shown