Distribution and localization of calmodulin-binding proteins in bull spermatozoa.
Previous studies from our laboratory have shown that a decrease in the calmodulin binding properties of a few sperm proteins occurs during the capacitation process, an effect associated with a decrease in intracellular calmodulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay assay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, whereas p30 is found in the fraction enriched with sperm heads. This latter calmodulin binding protein, p30, appears to be associated with the perinuclear theca. None of these binding proteins was solubilized by nonionic detergents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 was extracted only in conditions reported to isolate the perinuclear theca. Cellular localization of calmodulin binding proteins was also achieved by incubating spermatozoa fixed on slides with biotinylated calmodulin and revealed in a further step by fluorescein-conjugated streptavidin. Using this procedure, it was found that calmodulin binds to the sub- and postacrosomal areas of the sperm head along with the midpiece in the presence of Ca(2+). Only a sharp band of fluorescence at the subacrosomal area was observed when this procedure was performed in the absence of Ca(2+) in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when spermatozoa were incubated under capacitating conditions, in the presence of heparin, in agreement with the published effect of capacitation on calmodulin binding proteins.
Leclerc
P
P
Endocrinologie de la Reproduction, Centre de Recherche du CHUQ and Centre de Recherche en Biologie de la Reproduction, Quebec, Quebec, Canada G1L 3L5. pierre.leclerc@crsfa.ulaval.ca
Goupil
S
S
D000177:Acrosome / Q000502:physiology; D000818:Animals; D019921:Biotinylation; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003101:Collodion; D003902:Detergents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006493:Heparin / Q000494:pharmacology; D008297:Male; D013075:Sperm Capacitation; D013082:Sperm Tail / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000737:chemistry
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002614:Chelating Agents; D003902:Detergents; D005456:Fluorescent Dyes; D004533:Egtazic Acid; D003101:Collodion; D006493:Heparin