Evidence for the participation of epididymosomes in the association of “DE” with spermatozoa

MALDERA, JA; FORNÉS, MW; CUASNICÚ, PS

Buenos Aires, Argentina

Congreso; VIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología (SAB); 2006

Sociedad Argentina de Biología (SAB)

Rat protein DE, syntethized by the epididymal epithelium, associates with the sperm surface during sperm maturation. Sequential protein extraction from epididymal sperm revealed the existence of two populations of DE on sperm: a loosely bound population released during capacitation, and a strongly bound population involved in gamete fusion. While the loosely-bound population is associated with sperm by electrostatic interactions, the mechanism that anchors the tightly-bound DE to sperm remains unknown. Recent evidence shows that membranous vesicles present in the epididymal fluid, named epididymosomes (Ep), are involved in the transfer of some epididymal proteins to sperm. To investigate the participation of Ep in the transfer of the strongly-bound DE to sperm, Ep were isolated by ultracentrifugation of rat epididymal fluid and examined by electron microscopy to confirm their correct isolation. Western blot (Wb) analysis using anti-tubulin as a sperm marker and a specific antibody against DE, revealed both the absence of sperm and the presence of DE in the vesicle preparation. To examine the anchoring of DE to the Ep, the vesicles were subjected to different extraction treatments and the presence of DE in the protein extracts was analyzed by Wb. As previously observed for DE on sperm,  PBS, 2M NaCl, low pH(3) and 5U/ml PLC-PI (phospholipase C specific for inositol) were unable to remove DE from the Ep,  while high pH(11), 250mM DTT and 1% Triton X-100 completely extracted the protein from the vesicles. These results indicate the existence of a population of DE strongly bound to Ep, supporting the involvement of these epididymal vesicles in the transferring of DE to sperm during epididymal maturation.