Under both conditions (directly washed or after percoll gradient), the last pellet was directly extracted with an equal volume of nonreducing Laemmli sample buffer and centrifuged again; the supernatant was boiled after addition of beta-mercaptoethanol (2%) and is referred to as the sperm extract.
Cytoplasmic droplets were purified from the top layer of the discontinuous percoll gradient from the sperm samples of zones 8 and 9. This suspension was diluted in PBS, centrifuged at 15 000 X g, and the pellet resuspended and loaded on a 10-90% percoll gradient. The top white band separated after centrifugation was recovered. The purity of the droplets was assessed by microscopy after two successive washes in PBS (15 000 X g, 10 min, 4°C) and also by electron microscopy. This preparation was highly enriched in droplets, with very few contaminating sperm.
Each experiment was repeated at least twice with samples from two different animals. The most representative results are shown.
The 15 000 X g supernatant from the caudal fluid was ultracentrifuged at 48 000 X g (JA18; Beckman, Villepinte, France) or 100 000 X g (SW21; Beckman) for 2 h at 4°C (no major differences in the protein pattern of vesicles were observed between the two procedures by SDS-PAGE; see also ).