Distribution and localization of calmodulin-binding proteins in bull spermatozoa

Previous studies from our laboratory have shown that a decrease in the calm odulin binding properties of a few sperm proteins occurs during the capacit ation process, an effect associated with a decrease in intracellular calmod ulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay a ssay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, wh ereas p30 is found in the fraction enriched with sperm heads. This latter c almodulin binding protein, p30, appears to be associated with the perinucle ar theca. None of these binding proteins was solubilized by nonionic deterg ents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 wa s extracted only in conditions reported to isolate the perinuclear theca. C ellular localization of calmodulin binding proteins was also achieved by in cubating spermatozoa fixed on slides with biotinylated calmodulin and revea led in a further step by fluorescein-conjugated streptavidin. Using this pr ocedure, it was found that calmodulin binds to the sub- and postacrosomal a reas of the sperm head along with the midpiece in the presence of Ca2+. Onl y a sharp band of fluorescence at the subacrosomal area was observed when t his procedure was performed in the absence of Ca2+ in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when sperm atozoa were incubated under capacitating conditions, in the presence of hep arin, in agreement with the published effect of capacitation on calmodulin binding proteins.