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Fig. 6 | BMC Biology

Fig. 6

From: Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions

Fig. 6

Analysis of the involvement of DNM1 in epididymosome-sperm interaction. a Spermatozoa were incubated with biotin (membrane impermeant) labeled epididymosomes for 1 h before being subjected to immunofluorescence detection of biotin (green) and DNM1 (red). Representative immunofluorescence images of the different sperm labeling patterns detected after this period of co-incubation, and a schematic model, are presented to illustrate an apparent relocation of endogenous sperm DNM1 to the post-acrosomal domain and an accompanying transfer of biotinylated epididymosome proteins to an equivalent region. b To preclude the possibility that these changes in the localization reflected an unmasking of an additional pool of DNM1 due to spontaneous loss of the acrosomal domain, triple immunofluorescence staining was applied to detect DNM1 (yellow), biotin (red), and the outer acrosomal membrane (PNA; green) in the same cells. c The relative abundance of DNM1 was quantified by immunoblotting of sperm homogenates in naïve cells (Sperm only) as well as those exposed to co-culture with epididymosomes (Sperm + ES). Band intensity was normalized relative to that of α-tubulin, with the sperm only control nominally set to a value of 1 (n = 3). Individual data points for each replicate are provided in Additional file 7: Raw data. df Immunoelectron TEM was utilized to localize DNM1 in spermatozoa within the lumen of caput epididymal tissue. d A representative image is shown, with e the inset focusing on a site in which epididymosome-sperm docking was apparent (i.e., boxed region in panel d). Such interactions were predominantly found in association with the membrane overlying the post-acrosomal sheath/posterior region of the caput sperm head and invariably, gold labeling depicting the localization of endogenous sperm DNM1 was detected in the vicinity of the epididymosome docking sites (white arrows). f The specificity of gold labeling was confirmed by the inclusion of secondary antibody only controls, which consistently failed to label spermatozoa or epididymosomes. N, nucleus; Ac, acrosomal domain; ES, epididymosome

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