SCs secrete CD63-GFP–positive, exosome-like puncta. (A–D) Paired accessory glands (AG) expressing CD63-GFP in SCs connect to the ejaculatory duct (ED; A). Images of different magnifications show surface sections of SCs within the AG epithelium (B; from square in A, arrows mark two SCs), a transverse section through the lumen (C; bracketed region in B, arrows mark two SCs), and CD63-GFP–positive puncta in the lumen (D; square in C; an image at higher confocal gain setting is shown in Fig. S1 F). (E and F) High magnification images of surface (E) and transverse (F; asterisk marks the lumen containing GFP puncta) sections through the SC show that CD63-GFP is also found at the apical plasma membrane (arrowhead; F) and the limiting membrane of SC vacuoles, the majority of which have a dense ANCE-positive core (highlighted with arrows). One or two CD63-GFP–lined compartments per SC lack an ANCE-stained core but contain fluorescent puncta (E, asterisk). Fasciclin3 (Fas3) marks septate junctions at the cell surface, and DAPI marks nuclei (note that SCs and MCs are binucleate). (G and H) Vital staining of SCs with LysoTracker red reveals CD63-GFP–positive puncta (putative ILVs; arrows) in large acidic compartments (mMVBLs), distinguishing them from other compartment classes, such as the more abundant secretory vacuoles (SVs) and immature late endosomes (iLEs). All images show SCs from 3-d-old males incubated at 28.5°C after eclosion. (I) Schematic representation of compartments within SC in H; values below indicate the mean total numbers of each compartment in 6-d-old control SCs counted from multiple sections along the apical–basal axis; the numbers of SCs analyzed are given in brackets. Approximate outline of the SC is marked in E–H. Bars: (A and B) 200 nm; (C) 20 µm; (D–H) 5 µm.