Association of CRISP1 with sperm during epididymal maturation

MALDERA JA; VASEN G; COHEN DJ; ERNESTO JI; WEIGEL MUÑOZ M; CUASNICU PS

Barcelona, España

Congreso; 9th International Congress of Andrology; 2009

International Society of Andrology

Rat protein CRISP1 (Cysteine Rich Secretory Protein 1) is synthesized by the epididymis, and associates with the sperm surface during maturation. Observations from our laboratory support the existence of two populations of CRISP1 on sperm: one loosely-associated that is released during capacitation, and one strongly bound which remains after capacitation and participates in fertilization. However, the mechanisms involved in the association of CRISP1 with sperm remain mostly unknown. Considering that Zn2+ binds to cysteine-rich proteins and is present in high concentrations in the epididymis, in the present work we investigated the participation of this cation in the association of CRISP1 with sperm. Caput epididymal sperm were incubated in vitro for 3 hs with cauda epididymal fluid (free of sperm and membrane vesicles), in the presence of Zn2+ (0-2 mM), and the association of CRISP1 with the cells was analyzed by Western Blot. CRISP1 was detected in those sperm exposed to Zn2+ but not in those incubated in the absence of the cation or in the presence of Mg2+ or Zn2+ added with EDTA. The same results were obtained using purified CRISP1. Results also revealed that the in vitro-associated protein can be removed from sperm by exposure to EDTA or to NaCl. Finally, studies using cauda sperm indicated that EDTA removes CRISP1 from fresh sperm and that the presence of Zn2+ during capacitation inhibits CRISP1 release during this process. Altogether, these results support the involvement of Zn2+ in the association of the loosely-bound population of CRISP1 with sperm during epididymal maturation.