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(169) IMMUNODETECTION OF Ca2+-ATPases IN HUMAN SPERMATOZOA. Dorval, Véronique1, Leclerc, Pierre1, 1 Centre de Recherche en Biologie de la Reproduction, Québec, Canada ABSTRACT- The ability of spermatozoa to undergo an acrosome reaction and to fertilize the egg is gained during a final maturational event called capacitation. Many membranous and biochemical modifications characterize this process, one of which being an increase in the intracellular free Ca2+ concentration. Different experimental evidences support that the acrosome, a Golgi-derived structure, is an intracellular Ca2+ store. A number of studies have demonstrated the presence of Ca2+-ATPase activity in sperm head, suggesting their implication in maintaining a low intracellular free Ca2+ concentration. The aim of the present study was to investigate the presence of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) in human spermatozoa. Western blot analysis using a monoclonal antibody directed against SERCA 2 (recognizes both 2a and 2b isoforms) and a polyclonal antibody specific for SERCA 2 (recognizes both SERCA 2 and 3) revealed a 116 kDa band in a human sperm proteins extract, whereas no specific band was obtained using an anti-SERCA 3 monoclonal antibody. The protein recognized by the SERCA antibodies was extracted by the non-ionic detergent Triton X-100. Further experiments on the subcellular localization of the immunoreactive protein revealed that it is present in the membrane fraction following sonication or nitrogen cavitation of human spermatozoa. Indirect immunofluorescence studies using the monoclonal or polyclonal antibodies showed that SERCA 2 are localized at the acrosomal level. Further experiments are ongoing to provide more informations on the localization of Ca2+-ATPases and their possible involvement during capacitation. Supported by FCAR (VD), FRSQ (PL) and CIHR (PL). KEY WORDS: Ca2+-ATPase, human spermatozoa, acrosome, membrane |
