title: Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone.
abstract: Human spermatozoa stimulated with progesterone (a product of the cumulus and thus encountered by sperm prior to fertilization in vivo) apparently mobilize Ca(2+) and respond very differently according to the way in which the steroid is presented. A progesterone concentration ramp (0-3 microM) induces [Ca(2+)](i) oscillations (repetitive store mobilization) which modify flagellar beating, whereas bolus application of micromolar progesterone causes a single large transient (causing acrosome reaction) which is apparently dependent upon Ca(2+) influx. We have investigated Ca(2+)-mobilization and functional responses in human sperm exposed to 3 muM progesterone. The [Ca(2+)](i) response to progesterone was abolished by 4 min incubation in 0 Ca(2+) medium (2 mM EGTA) but in nominally Ca(2+)-free medium (no added Ca(2+); 0 EGTA) a smaller, slow response occurred. Single cell imaging showed a similar effect of nominally Ca(2+)-free medium and approximately 5% of cells generated a small transient even in the presence of EGTA. When cells were exposed to EGTA-containing saline (5 min) and then returned to nominally Ca(2+)-free medium before stimulation, the [Ca(2+)](i) transient was greatly delayed (approximately 50 s) and rise time was doubled in comparison to cells not subjected to EGTA pre-treatment. We conclude that mobilization of stored Ca(2+) contributes a 'slow' component to the progesterone-induced [Ca(2+)](i) transient and that incubation in EGTA-buffered saline is able rapidly to deplete this store. Analysis of flagellar activity induced by 3 muM progesterone showed an effect (modified beating) associated with the [Ca(2+)](i) transient, in >80% of cells bathed in nominally Ca(2+)-free medium. This was reduced greatly in cells subjected to 5 min EGTA pre-treatment. The store-mediated transient showed a pharmacological sensitivity similar to that of progesterone-induced [Ca(2+)](i) oscillations (consistent with filling of the store by an SPCA) suggesting that the transient induced by micromolar progesterone is a 'single shot' activation of the same store that generates Ca(2+) oscillations.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: K
authors.initials: K
authors.lastname: Bedu-Addo
authors.forename: C L R
authors.initials: CL
authors.lastname: Barratt
authors.forename: J C
authors.initials: JC
authors.lastname: Kirkman-Brown
authors.forename: S J
authors.initials: SJ
authors.lastname: Publicover
mesh_terms: D000177:Acrosome / Q000378:metabolism; D020101:Acrosome Reaction; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D011374:Progesterone / Q000378:metabolism* / Q000494:pharmacology; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D011374:Progesterone; D004533:Egtazic Acid; D019284:Thapsigargin; D002118:Calcium