This result thus confirmed that these proteins are mainly associated with the vesicles.
Presence of Epididymal Hydrophobic Proteins Within the Vesicles
To investigate whether hydrophobic proteins cosediment with the vesicles, we used a similar immunoblotting approach (Fig. 5). The first protein sought was the 17-kDa hydrophobic protein that has previously been described in rams.
This protein is secreted exclusively by the epithelium of the cauda region (zones 7-9) and transferred to the sperm membrane. The Western blot showed that this protein was linked to the vesicles but that a large part also remained in the fluid after centrifugation.
Because PEBP (phosphatidyl-ethanolamine binding protein) was found by mass spectrometry to be associated with the vesicles, we analyzed whether other proteins that bind and transport hydrophobic compounds in epididymal fluid were also present. One isoform of the retinol acid binding protein (E-RABP) was found to be present in the vesicles as well as prostaglandin D2. In contrast, the cholesterol transport protein (HE1/NP-C2/CTP) was not found to be associated with the vesicles.