(a) Prior to detection of DNM1 and DNM2 proteins, aldehyde/sulphate latex beads were used to concentrate caput epididymosomes amenable with downstream fluorescence imaging applications. (TIF 1173 kb) 12915_2019_653_MOESM2_ESM.tif (1.1M) GUID:?6CC08F4B-6288-4021-B61D-D9B88A862242 Additional file 3: Figure S3. Detection of GM1 labeling of mouse caput epididymal spermatozoa. Immunofluorescence detection of GM1 gangliosides (an abundant lipid raft marker) was facilitated by labeling of caput epididymal spermatozoa with Alexa Fluor 594 conjugated cholera toxin B subunit. (aCe) A myriad of fluorescence staining patterns for GM1 were observed using confocal microscopy and the dominant profiles are depicted. (TIF 562 kb) 12915_2019_653_MOESM3_ESM.tif (562K) GUID:?E7AB511D-68EE-4EDC-A9AC-6B495FE04BE2 Additional file 4: Table S1. Details of antibodies used throughout this study (DOCX 23 kb) 12915_2019_653_MOESM4_ESM.docx (24K) GUID:?A4134775-B791-41FC-A075-AC859685E42B Additional file 5: Figure S4. Assessment of epididymosome purity. A suite of assays were employed to assess the enrichment Oxybenzone of epididymosomes, including (a) quantitative Ntn2l assessment of the protein content in the 12 equal fractions recovered after density ultracentrifugation; (b) immunoblotting to detect the distribution of the epididymosome marker FLOT1 within each of the 12 fractions; (c) detection of FLOT1 in epididymosomes concentrated via adhesion to aldehyde/sulphate beads; and (d) TEM assessment of the ultrastructure of the epididymosome population isolated from the pooling of fractions 9 and 10. Based on this analysis, epididymosomes partitioning into fractions 9 and 10 were pooled and used throughout the reported studies. (TIF 1607 kb) 12915_2019_653_MOESM5_ESM.tif (1.5M) GUID:?5140FBC5-CF44-4CDB-A623-CE3A620E08DD Additional file 6: Table S2. Excitation and emission wavelengths used for detection of the different combinations Oxybenzone of fluorophores in this study. (DOCX 13 kb) 12915_2019_653_MOESM6_ESM.docx (13K) GUID:?44A070FE-F6A1-4F7B-8DF1-AC704A1F1EC8 Additional file 7: Raw data. This file contains raw data with individual data points or replicates for Figs. ?Figs.6,6, ?,7,7, ?,9,9, and ?and1010 (i.e., those experiments in which is similar to DNM1; endogenous DNM2 was also readily detected in the peri-acrosomal domain of caput spermatozoa (Fig.?7a). However, the DNM2 isoform did not appear to undergo any pronounced change in location upon co-incubation with epididymosomes (Fig. ?(Fig.7a).7a). In this context, only weak DNM2 labeling was observed in the post-acrosomal domain coinciding with those cells in which a substantial amount of biotinylated protein transfer was detected; raising the prospect that this additional pool of DNM2 may have been transferred to the cells as part of the epididymosome cargo. However, despite the detection of DNM2 within caput epididymosomes (Additional?file?2: Figure S2), densitometric analysis revealed only a modest, non-significant, increase in the abundance of DNM2 before and after epididymosome co-incubation (Fig.?7b). Open in a separate window Fig. 7 Analysis of the involvement of DNM2 in epididymosome-sperm interaction. a Spermatozoa were incubated with biotin Oxybenzone (membrane impermeant)-labeled epididymosomes for 1?h before being subjected to immunofluorescence detection of biotin (green) and DNM2 (red). Representative immunofluorescence images of the different sperm labeling patterns detected after this period of co-incubation are provided to illustrate the labeling of DNM2 in the acrosomal domain and minimal co-localization with transferred biotinylated proteins. Indeed, only relatively weak DNM2 labeling was detected in the post-acrosomal domain of those cells that incorporated abundant biotinylated proteins. b The relative abundance of DNM2 was quantified by immunoblotting of sperm homogenates in na?ve cells (Sperm only) as well as those exposed to co-culture with epididymosomes (Sperm + ES). For the purpose of comparing the relative abundance of DNM2, band intensity was normalized relative to that of -tubulin, with sperm only control nominally set to a value of 1 1 ( em n /em ?=?3). Individual data points for each replicate are provided in Additional?file?7: Raw data. Oxybenzone These experiments were replicated three different times with each sample representing pooled material obtained from at least three mice.

(a) Prior to detection of DNM1 and DNM2 proteins, aldehyde/sulphate latex beads were used to concentrate caput epididymosomes amenable with downstream fluorescence imaging applications