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Signaling and Signal Transduction in Endocrine Tissues (M748) IDENTIFICATION AND LOCALIZATION OF SERCA 2 IN MAMMALIAN SPERM. Lawson, Christine1, Leclerc, Pierre1, 1 Université Laval, Centre de Recherche du CHUQ, Quebec, QC, Canada ABSTRACT- The acrosome reaction is a calcium-dependent exocytotic event triggered by the binding of the spermatozoa to the zona pellucida. During this process there is an important rise in [Ca2+]i du to Ca2+ depletion from internal stores accompanied by a massive Ca2+ influx. Previous reports have shown that the acrosome can act as a Ca2+ store and that depletion of thapsigargin-sensitive stores induces acrosome exocytosis in capacitated spermatozoa from different mammalian species.Therefore, the effect of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA), suggests the presence and the implication of SERCA in the active Ca2+ uptake during mammalian sperm capacitation. The objective of this present study was to localize and identify a SERCA pump in mammalian sperm. By western blot, we identified SERCA 2 in murine, bovine and human sperm. Two different bands were found in each species with masses ranging from 105 kDa to 115 kDa which may correspond to the isoforms SERCA 2a, 2b and 2c described in the literature. Futhermore, in human sperm fractionnated into subcellular fractions, the two isoforms were detected in the acrosomal membrane (110 kDa) and in the fraction containing the heads with their midpiece attached (105 kDa). Localization by indirect immunofluorescence revealed the presence of SERCA 2 in the acrosome region and midpiece of human sperm whereas murine and bovine sperm only showed SERCA 2 expression in the acrosomal region. Immunohistochemistry on human testis revealed the expression of SERCA 2 starting at the stage of spermatocytes. This study is the first to identify a SERCA pump in mammalian spermatozoa. This study is supported by the Canadian Institute of Health Research. KEY WORDS: sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, intracellular calcium, acrosome reaction, mammalian sperm |
