SC exosomes interact with sperm in females. (A) Schematic of female reproductive tract in D showing mating plug (mp), uterus (ut), oviduct (ovi), and sperm storage organs (spermathecae [spt] and seminal receptacle [sr]) as well as the autofluorescent hindgut (HG). (B and C) w¹¹¹⁸ female reproductive tract dissected 20 min after mating to either a control w¹¹¹⁸ (B) or SC>CD63-GFP (C) male, imaged under identical confocal settings. Images show lumen of the female uterus (asterisks) containing sperm whose heads are visible with DAPI staining (arrowheads). (D and E) Female reproductive tracts 20 min after mating to a protamineB-RFP; SC>CD63-GFP male. (D) ProtamineB-RFP (prot-RFP), which marks sperm heads, accumulates in the anterior uterus (red arrowhead) and oviduct (red arrow). Most CD63-GFP–positive exosomes localize to the posterior uterus (green asterisk), but they can also be seen in the anterior uterus (green arrowhead) and oviduct (green arrow). The autofluorescent mating plug (closed asterisk) is shown. (E) CD63-GFP–positive exosomes colocalize with sperm heads marked by protamineB-RFP (open arrows). (F) Orthogonal view of a z stack through near-complete ring of CD63-GFP fused to ProtamineB-RFP sperm head (arrows). The confocal image below the blue line shows part of the female reproductive tract lumen. The two images above the blue line, separated by a white line, are green (top) and red/green (bottom) z stacks captured in the z plane marked in the bottom image. Note multiple pairs of parallel GFP-positive lines (arrowheads), potentially produced by sperm tail fusion. (G) These (arrowheads) and irregular extended lines of fluorescence (arrow) are also seen in G, which shows a maximum 3D projection image of a z stack from a female anterior uterus and oviduct where the epithelium is marked by actin>CD8-RFP (also shown in Video 3). (H) Female reproductive tracts were dissected and fixed at specific times after the start of mating to SC>CD63-GFP males. The frequency of exosome–sperm interaction events within the female reproductive tract was analyzed (n = 8 for each time point after the start of mating [ASM]). Only fusions to the sperm heads are included. Bars: (B and C) 50 µm; (D) 200 µm; (E and F) 5 µm; (G) 20 µm.