MOLECULAR MECHANISMS INVOLVED IN THE ASSOCIATION OF CRISP1 TO SPERM DURING EPIDIDYMAL MATURATION.

VASEN G; MALDERA JA; COHEN DJ; CUASNICU PS

Congreso; Fifth International Conference on Epididymis.; 2010

Cysteine-Rich Secretory Protein 1 (CRISP1) is synthesized in the rat epididymis and associates with the sperm surface during maturation. Our previous results support the existence of two populations of CRISP1 in sperm: one loosely-associated that is released during capacitation, and one strongly bound which remains after capacitation and participates in fertilization. However, the mechanisms underlying the association of these populations remain unknown. Considering the high concentrations of zinc (Zn2+) in the epididymal lumen, in the present work we investigated the potential involvement of this cation in the association of CRISP1 to sperm during maturation.  For this purpose, caput epididymal sperm were incubated with biotinylated CRISP1 in the presence or absence of 1mM Zn2+ evaluating the association of the protein to the cells by flow cytometry. Results revealed that only the cells exposed to the cation exhibited an increase in fluorescence that was dependent on CRISP1 concentration and could be blocked by EDTA. To examine the localization of the bound protein on the cells, both caput and cauda sperm exposed to biotinylated CRISP1 in the presence or absence of  Zn2+ were analyzed by epifluorescence microscopy using avidin-FITC. While a faint labeling was observed in the absence of Zn2+, caput and cauda sperm exposed to the cation exhibited a clear staining in the tail and in both the tail and the head, respectively, suggesting differences in the ability of the sperm plasma membrane to associate CRISP1 during maturation. No changes in CRISP1 tryptophan fluorescence spectrum were detected when the protein was exposed to increasing Zn2+ concentrations indicating that the presence of the ion would not affect CRISP1 conformation. To evaluate the potential formation of CRISP1 complexes induced by the presence of Zn2+, the epididymal protein was incubated with the cation in vitro and then subjected to native gel electrophoresis and Western blot using the polyclonal antibody anti-CRISP1. Under these conditions, a high molecular weight complex was detected. To investigate the possible formation of such a complex in vivo, epididymal fluids were analyzed by electrophoresis in native gels and Western blot. Results revealed the presence of a high molecular weight band not detected in fluids pre-treated with EDTA. When the high molecular weight band was extracted from the nitrocellulose membrane by SDS and re-analyzed by SDS-PAGE and Western blot, a band of 32kDa was detected confirming the presence of CRISP1 in the fluid complex. Altogether, these results suggest that a molecular complex between CRISP1 and Zn2+ might be involved in the in vivo association of CRISP1 to sperm during epididymal maturation. Keywords:   CRISP1, Zinc, sperm, epididymis, maturation Financial Support: National Research Council of Argentina grant, National Agency of Scientific and Technological Promotion grant and World Health Organization (WHO) RMG grant