Alpha-L-fucosidase of epididymal fluid is active on bull spermatozoa

AGUILERA AC; ELÍAS-RODRÍGUEZ P; CARVELLI L; SOSA MA; BOSCHÍN V; ROBINA I

Campos do Jordao

Simposio; VI International Symposium on Animal Biology of Reproduction (ISABR2016); 2016

Brazilian College of Animal Reproduction (CBRA)

The mammalian epididymis plays an important role in the acquisition of motility and fertilizing ability ofspermatozoa. During passage through the epididymis, spermatozoa undergo biochemical and morphologicalchanges, resulting in a gamete competent for fertilization. These changes include modifications of glycoconjugates on the cell surface as the result of sequential interactions with proteins secreted by the epithelium along the epididymal duct. Among the several proteins secreted by the epididymal epithelium, glycosidases have been found in the epididymal lumen of domestic animals, such as bulls. Alpha-L-fucosidase (FUC) appears to be important for the fertilizing ability of the spermatozoa, since, high levels of this enzyme have been found in the epididymal fluid of bulls with high fertilizing capacity. Regarding this, we proposed to study the secreted FUC of bull cauda epididymis and correlate with fucosylated status of spermatozoa. We incubated spermatozoa from caput or cauda with FITC-conjugated UEA-I lectin and analyzed the fucosylation by fluorescence miscroscopy and flow cytometry. It was observed a strong post-acrosomal detection in spermatozoa from the caput, which is much lesser in cauda spermatozoa. This change was significant as judged by flow cytometry quantification. Moreover, we evaluated the pattern of protein fucosylation, employing biotin-conjugated UEA-I after blotting onto nitrocellulose membranes. Consistent with the previous results, we observed a decrease of fucosylation in several proteins of cauda spermatozoa, although an increase was observed in other proteins. This decreased fucosylation may be related to an increased activity of FUC in the lumen of cauda epididymis. By western blot, using anti-FUC, we found that the enzyme is expressed in the tissue of caput, corpus and cauda, although only is secreted in the cauda epididymis. These results were confirmed evaluating the enzyme activity by spectrofluorometry. In order to determine if FUC from epididymal lumen is responsible of fucose removal in the sperm surface, we performed an assay in which a crude cauda fluid was incubated with caput spermatozoa. After incubation we observed (by flow cytometry) a significant decrease of fucose detection which was reversed with synthetic specific inhibitors for FUC. In conclusion, we provide direct evidence that FUC from epididymal fluid participates in removing fucose from spermatozoa, as a step of sperm maturation in bull epididymis