Patterns of [Ca2+]i mobilization and cell response in human spermatozoa exposed to progesterone

Human spermatozoa stimulated with progesterone (a product of the cumulus and thus encountered by sperm prior to fertilization in vivo) apparently mobilize Ca2+ and respond very differently according to the way in which the steroid is presented. A progesterone concentration ramp (0-3 μM) induces [Ca2+]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 Ca2+ influx. We have investigated Ca2+-mobilization and functional responses in human sperm exposed to 3 μM progesterone. The [Ca2+]i response to progesterone was abolished by 4 min incubation in 0 Ca2+ medium (2 mM EGTA) but in nominally Ca2+-free medium (no added Ca2+; 0 EGTA) a smaller, slow response occurred. Single cell imaging showed a similar effect of nominally Ca2+-free medium and ≈ 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 Ca2+-free medium before stimulation, the [Ca2+]i transient was greatly delayed (≈ 50 s) and rise time was doubled in comparison to cells not subjected to EGTA pre-treatment. We conclude that mobilization of stored Ca2+ contributes a 'slow' component to the progesterone-induced [Ca2+]i transient and that incubation in EGTA-buffered saline is able rapidly to deplete this store. Analysis of flagellar activity induced by 3 μM progesterone showed an effect (modified beating) associated with the [Ca2+]i transient, in > 80% of cells bathed in nominally Ca2+-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 [Ca2+]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 Ca2+ oscillations.