Evidence for the Involvement of Zinc in the Association of CRISP1 with Sperm During Epididymal Maturation in Rats.

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

BIOLOGY OF REPRODUCTION

SOC STUDY REPRODUCTION

Lugar: Oregon; Año: 2011 vol. 85 p. 503 - 510

0006-3363

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm duringmaturation and participates in fertilization. Evidence indicates the existence of two populationsof 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 spermremain mostly unknown. Considering the high concentrations of Zn2+ 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 Zn2+, andbinding of CRISP1 to sperm analyzed by Western blot. An increase in CRISP1 was detected insperm exposed to Zn2+ but not if the cation was added with EDTA. The same results wereobtained using purified CRISP1. CRISP1 association with sperm was dependent on epididymalfluid and Zn2+ concentrations, and incubation time. Treatment with NaCl (0.6 M) removed the invitro-bound CRISP1 indicating that it corresponds to the loosely-bound population. Flowcytometry analysis of caput sperm exposed to biotinylated CRISP1/avidin-FITC revealed thatonly the cells incubated with Zn2+ exhibited an increase in fluorescence. When these spermwere examined by epifluorescence microscopy, a clear staining in the tail accompanied by aweaker labeling in the head was observed. Detection of changes in the tryptophan fluorescenceemission spectra of CRISP1 when exposed to Zn2+ supported a direct interaction betweenCRISP1 and Zn2+. Incubation of either cauda epididymal fluid or purified CRISP1 with Zn2+,followed by native-PAGE and Western blot revealed the presence of high molecular weightCRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results supportthe involvement of CRISP1-Zn2+ complexes in the association of the loosely bound population ofCRISP1 with sperm during epididymal maturation.BOR Papers in Press. Published on May 18, 2011 as DOI:10.1095/biolreprod.111.091439Copyright