Evaluación de la presencia y localizacion de Disadherina en espermatozoides humanos en diferentes estadíos funcionales de la gameta.

GABRIELLI, VAZQUEZ-LEVIN ET AL (VER DETALLE EN RESUMEN)

Academia Nacioal de Medicina. Buenos Aires, Argentina

Congreso; V Congreso Argentino de Andrologia; 2007

Sociedad Argentina de Andrologia

Resumen en ingles EXPRESSION OF DYSADHERIN IN THE MALE REPRODUCTIVE TRACT, AND PROTEIN IMMUNODETECTION IN HUMAN SPERMATOZOA. 1GABRIELLI N., 1MATOS M.L., 1GONGORA A., 1BALDI A., 2HIROHASHI S., 2NAKANISHI Y., 1VAZQUEZ-LEVIN, MH. 1Instituto de Biología y Medicina Experimental. Buenos Aires, Argentina. 2National Cancer Center Research Institute, Tokyo, Japan.   Introduction: Dysadherin (dys) is a transmembrane glycoprotein (50-55 KDa) initially identified in tumoral tissues, and found in some normal cell lines. Dys would modulate epithelial cadherin (Ecad) and Na,K-ATPase functions (Nam et al, 2007); the expression of Ecad and the Na,K pump in the human spermatozoon has been previously reported (Vazquez-Levin et al, 2003; Sanchez et al, 2006, respectively). Objective: the aim of this study was to determine the expression of dys in human male reproductive tissues and gametes (RNA and protein forms), and to evaluate its localization in non-capacitated, capacitated and acrosome-reacted (AR) spermatozoa. Methods: The expression of dys RNA in testis, epididymis and spermatozoa was evaluated by reverse transcription of total RNA coupled to PCR analysis (RT-PCR). Presence of the dys protein was assessed by SDS-PAGE and Western Immunoblotting (WIB), and its localization in the gamete was analyzed by fluorescence immunocytochemistry in co-localization studies using an anti dys monoclonal antibody (NCC-M53) in combination with cell staining with pisum sativum agglutinin (FITC-PSA) to determine the sperm acrosomal status. MDA-MB-231 and HUVEC cells were used as controls. Results: RT-PCR studies revealed the expression of dys in testis, epididymis (caput, corpus and cauda) and spermatozoa. Immunocytochemical analysis showed dys localization in the principal piece of the sperm flagellum in more than 90% of non-capacitated, capacitated and reacted spermatozoa (n=5 donors); in addition, permeabilized spermatozoa had a specific signal for dys in the acrosome (85 ± 6 % of cells), that disappeared in AR-cells. WIB of protein sperm extracts revealed a dys form of higher Mr (91 KDa) than expected, possibly caused by changes in protein glycosylation. Conclusions: This is the first report describing the expression of dysadherin in human spermatozoa. Dysadherin is immunolocalized in subcellular regions that participate in sperm motility (flagellum) and gamete interaction (acrosome) during fertilization, and in which the presence of Na,K-ATPase and Ecad has been previously reported.