Evidence for the involvement of zinc in the association of crisp1 with sperm during epididymal maturation

Abstract

Rat epididymal protein CRISP1 (Cysteine-Rich Secretory Protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound, released during capacitation, and one strongly bound, that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zinc present in the epidydimis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were co-incubated with epididymal fluid in the presence or absence of Zinc and binding of CRISP1 to sperm analyzed by Western blot. An increase in CRISP1 was detected in sperm exposed to Zinc but not if the cation was added with EDTA. The same results were obtained using purified CRISP1. CRISP1 association with sperm was dependent on epididymal fluid and concentrations, and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1 indicating that it corresponds to the loosely-bound population. Flow cytometry analysis of caput sperm exposed to biotinylated CRISP1/avidin-FITC revealed that only the cells incubated with exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail accompanied by a weaker labeling in the head was observed.Both the change in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zinc and the detection of two potential binding sites in a modeled CRISP1 three-dimensional structure, supported the interaction between CRISP1 and Zinc. Incubation of either cauda epididymal fluid or purified CRISP1 with Zinc followed by native-PAGE and Western blot revealed the presence of high molecular weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results suggest that CRISP1-Zinc omplexes would be involved in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.