We therefore analyzed the behavior of other vesicle proteins. We observed that the amounts of E-NNP3, neprilysin, lactadherin-PAS 6/7, 35-kDa G-beta in zone 7 were strongly decreased after high-speed centrifugation (Fig. 6C), confirming that these proteins are associated with the vesicles as soon as they are present in the fluid.
Origin(s) of the Vesicles
Because we have previously observed that 5-10% of sperm died during epididymal transit and because sperm lose their cytoplamic droplet during the corpus transit, we checked whether some of the vesicles were derived from the sperm cells or their droplets. For this, we used different antibodies to investigate whether the vesicle proteins were also present on the sperm and/or the cytoplasmic droplet.
Sperm obtained from the caput region (zone 2) that retain their cytoplasmic droplets; from the corpus region (zone 6), where sperm with or without droplets coexist; from the cauda epididymis (zones 8-9), where sperm have lost their droplets were washed by percoll gradient centrifugation before extraction. This also purified intact cytoplasmic droplets released in the caudal fluid.