Fig. 4

Examination of the transfer of lipophilic dye (PKH26) between epididymosomes and spermatozoa. a–g Caput spermatozoa were briefly co-cultured (≤ 5 min) with populations of epididymosomes preloaded with the lipophilic dye, PKH26. After incubation, spermatozoa were washed and fixed prior to the analysis of PKH26 labeling profiles via immunofluorescence microscopy. Representative immunofluorescence images of different spermatozoa confirmed the incorporation of PKH26 dye, with staining patterns appearing broadly similar to those documented for proteins labeled with membrane impermeant biotin. That is, PKH26-labeled lipids were predominantly transferred to the SAR and post-acrosomal domain of the sperm head. Representative controls were included in which spermatozoa were either h incubated with non-labeled epididymosomes to confirm no auto-fluorescence or alternatively, i directly labeled with PKH26 dye (independent of epididymosomes), which resulted in staining of the whole spermatozoon. j To examine the kinetics of PKH26 transfer, caput spermatozoa were co-cultured with PKH26-labeled epididymosomes and sampled at regular intervals during the course of a 3-h incubation. The dominant pattern of post-acrosomal labeling of the spermatozoa were quantified, with 100 cells being examined per sample (n = 3; graphical data are presented as mean ± SEM). **P < 0.01