CONICET | Buscador de Institutos y Recursos Humanos

Mammalian seminal plasma contains small membranous vesicles, called prostasomes (prostate derived vesicles) and epididymosomes (epididymis derived vesicles), that transfer proteins to the sperm plasma membrane. Our group previously described the presence of the cell-cell adhesion glycoprotein Epithelial Cadherin (E-cad) in human seminal plasma (hSP) and in ejaculated sperm, and confirmed previous evidence of its expression in the epididymis (Vazquez-Levin et al, 2003). The aim of this study was to evaluate the presence of E-cad protein forms in both soluble and membranous vesicle fractions of the hSP. Materials & Methods: hSP was obtained from normozoospermic donors (WHO, 1999). Membranous vesicles were isolated by ultracentrifugation of hSP at 100,000xg and further purification by gel filtration on Sephadex G-200. Protein extracts from total and ultracentrifugated hSP, from membranous vesicles recovered before and after gel filtration analysis, and from human sperm were prepared in Laemmli sample buffer and analyzed by SDS-PAGE and Western Immunoblotting using antibodies towards three different E-cad domains. Results: Four high molecular weight forms of E-Cad were detected in protein extracts from crude hSP and from purified membranous vesicles: the full length 122 KDa mature protein, a N-terminus truncated 105KDa form, and two protein forms of 97 KDa and 86 KDa lacking the cytplasmic region; similar E-cad protein patterns were previously described in human sperm (Lentz et al, 2003). The 86 KDa form was also immunodetected in the ultracentrifuged hSP and may correspond to the E-cad ectodomain previously described in other cell systems. Conclusion: E-cad protein forms are present in the soluble fraction as well as in the membranous vesicles purified from hSP. Immunoelectron microscopy analysis of the purified vesicles will further characterize E-cad localization.