IDENTIFICATION OF EPITHELIAL CADHERIN IN MEMBRANOUS VESICLES ISOLATED FROM HUMAN SEMINAL PLASMA

ZACCHI N; LENTZ E; VINCENTI A E; FORNES M W; VAZQUEZ M H

Chicago, Illinois

Congreso; Reunión Anual de la Sociedad Americana de Andrología; 2006

Sociedad Americana de Andrología

IDENTIFICATION OF epithelial CADHERIN IN MEMBRANOUS VESICLES ISOLATED FROM HUMAN SEMINAL PLASMA 1Zacchi N, 1Lentz E, 2Vincenti A, 2Fornes M, 1Vazquez-Levin MH. 1Instituto de Biología y Medicina Experimental (IBYME CONICET-UBA), Buenos Aires; Instituto de Histología y Embriología (IHEM-CONICET), Mendoza. Argentina (presented by Vazquez-Levin MH) 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 (Ecad) in human seminal plasma (hSP) and in ejaculated sperm, and confirmed its expression in the epididymis (Vazquez-Levin et al, 2003). The aim of this study was to evaluate the presence of Ecad protein forms in both soluble and membranous vesicle fractions of the hSP. 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. Transmission electron microscopy analysis confirmed the presence of small electron dense and slightly larger pale vesicles in the membranous fractions recovered before and after gel filtration analysis. Western immunoblotting of protein extracts from crude hSP and from purified membranous vesicles developed with three different antibodies towards Ecad (sc#7870, Sta Cruz; HECD-1, Zymed-Invitrogen; #C20820, Transduction BD) revealed the presence of four high Mr weight Ecad forms: the full length 122 KDa mature protein, a N-terminus truncated 105 KDa form, and two protein forms of 97 KDa and 86 KDa lacking the cytplasmic region. Similar Ecad 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. In conclusion, Epithelial cadherin is present in the soluble fraction as well as in “prostasome like” membranous vesicles purified from human seminal plasma. Immunoelectron microscopy analysis of the purified vesicles will further help characterization of E-cad localization. This study has been supported by grants from World Health Organization and the National Research Council (CONICET) of Argentina to MHVL.