Participation of epididymosomes in the association of epididymal protein CRISP1 with spermatozoa

MALDERA JA; FORNÉS MW; COHEN DJ; ERNESTO JI; VASEN G; CUASNICÚ PS

Philadelphia, USA

Congreso; 34th Annual Meeting of American Society of Andrology; 2009

American Society of Andrology, Supplemente March/April 2009, pag 46

Rat protein CRISP1 (Cysteine Rich Secretory Protein 1) associates with the sperm surface during epididymal maturation. Sequential protein extraction experiments revealed the existence of two populations of CRISP1 on epididymal sperm: a major population loosely associated with the cells that is released during capacitation, and a minor, strongly-bound protein, that remains on sperm and participates in fertilization. In spite of its relevance, the mechanisms that anchor the tightly-bound CRISP1 to sperm are still unknown. Recent evidence shows that membranous vesicles from the epididymal fluid named epididymosomes are involved in the association of proteins with the sperm surface during epididymal passage. In view of this, in the present work we investigated the participation of epididymosomes in the transfer of CRISP1 to sperm. For this purpose, epididymal vesicles were obtained by ultracentrifugation of rat epididymal fluid and then examined by electron microscopy to confirm their correct isolation. Western blot analysis using anti-tubulin and anti-CRISP1 as first antibodies revealed the absence of sperm in the epididymosomes preparation and the association of CRISP1 with the vesicles. In order to examine the anchoring of CRISP1 to the epididymosomes, the vesicles were subjected to different protein extraction treatments and the presence of CRISP1 in the protein extract was subsequently analyzed by Western blot. As previously observed for the protein bound to sperm, PBS, 2M NaCl, low pH(3) and 5U/ml PLC-PI (phospholipase C specific for inositol) were not capable of removing CRISP1 from the epididymosomes, while high pH(11), 250mM DTT (dithiothreitol) and 1% Triton X-100 completely extracted the protein from the vesicles. Together, these results confirmed the existence of a population of CRISP1 tightly-bound to the epididymosomes, supporting the participation of these vesicles in the mechanism by which CRISP1 strongly associates with sperm during epididymal transit.