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PRESENCE AND LOCALIZATION OF EPITHELIAL CADHERIN IN FROZEN-THAWED BULL SPERM IN DIFFERENT FUNCTIONAL STATES 1Caballero J., 2Cetica P., 2Dalvit G., 1Marin Briggiler C.,1Vazquez-Levin M.H. 1Instituto de Biolog¨ªa y Medicina Experimental (IBYME-CONICET); 2Biological Chemistry Area, School of Veterinary Medicine. University of Buenos Aires, Buenos Aires, Argentina. (Presented by Vazquez-Levin M.H) Epithelial cadherin (Ecad) is a Ca+2 dependent cell-cell adhesion glycoprotein. Its presence has been reported in human and murine sperm, and data suggest its participation in fertilization. The aim of the study was to evaluate the presence of Ecad in frozen-thawed bull sperm and to assess its localization under different experimental conditions that resemble functional changes that sperm undergo during fertilization. Frozen-thawed semen from 5 Holstein bulls of known fertility were used; evaluations were done in all samples after the following treatments: 1) Frozen-thawed sperm and semen extender removal with Sp-TALP medium, 2) Selection of motile sperm by glass wool column filtration, 3) Incubation of motile sperm in medium containing 60¦Ìg/ml heparin to promote capacitation (control same + 5 mM glucose) and 4) Incubation of capacitated sperm (cap-sperm) with 100 ¦Ìg/ml Lysophosphatidylcholine (LPC) to promote acrosomal exocytosis. Ecad localization was done by immunocytochemistry with an specific antibody (sc#7870; Sta Cruz Biotech). In all cases, % live sperm (Eosin Y staining), % progressive motile sperm (subjective evaluation), % capacitated sperm (CTC (ChlorTetraCycline) staining), and % acrosome intact sperm (FITC-Pisum Sativum Agglutinin (FITC-PSA) staining) was assessed. In frozen-thawed and selected motile sperm (21¡À2% cap-perm, 98¡À1% with intact acrosome; mean ¡À SEM), a strong signal for Ecad was observed in the apical ridge, accompanied with a weak staining in the acrosome and post-acrosomal regions (63¡À3%; 77¡À2%; respectively). Sperm incubated under capacitating conditions (57¡À1% cap-sperm, 87¡À1% with intact acrosome) remained immunoreactive to E-cad in the apical ridge (57¡À6%), although no staining was detected in the acrosome. LPC-acrosome reacted (44¡À2% acrosome reaction) sperm lost the E-cad signal over the apical ridge in all cases, and showed a signal in the sperm head, in regions that would suggest Ecad localization to the inner acrosomal/nuclear membrane (54¡À4%) and to the equatorial segment (21¡À5%). Conclusion: Epithelial cadherin is present in frozen-thawed sperm. Under all experimental conditions tested (non-capacitated,capacitated and acrosome reacted sperm), Epithelial cadherin was localized in sperm regions proposed to participate in the interaction with oviductal cells and oocyte envelopes. This study has been supported by grants to MHVL from the National Agency to Promote Science and Technology (ANPCyT) and from the National Research Council (CONICET) of Argentina.