Abstract
Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient of progesterone (0-3 microM) to simulate approach to the oocyte induced a slowly developing increase in [Ca(2+)](i) upon which, in many cells, slow oscillations were superimposed. [Ca(2+)](i) oscillations often started at very low progesterone (<10 nm), and their frequency did not change during the subsequent rise in concentration. Oscillations also occurred, but in a much smaller proportion of cells, in response to stepped application of progesterone (3 microM). When progesterone was removed, [Ca(2+)](i) oscillations often persisted or quickly resumed. Superfusion with low-Ca(2+) bathing medium (no added Ca(2+)) did not prevent [Ca(2+)](i) oscillations, but they could be abolished by addition of EGTA or La(3+). Inhibitors of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases or inositol trisphosphate signaling had no effect on [Ca(2+)](i) oscillations, but pharmacological manipulation of ryanodine receptors affected both their frequency and amplitude. Staining of live spermatozoa with BODIPY FL-X ryanodine showed localization of ryanodine binding primarily to the caudal part of the head and mid-piece. [Ca(2+)](i) oscillations did not induce acrosome reaction, but in cells generating oscillations, the flagellar beat mode alternated in synchrony with the oscillation cycle. Flagellar bending and lateral movement of the sperm head during [Ca(2+)](i) peaks were markedly increased compared with during [Ca(2+)](i) troughs. This alternating pattern of activity is likely to facilitate zona penetration. These observations show that progesterone initiates unusual and complex store-mediated [Ca(2+)](i) signaling in human spermatozoa and identify a previously unrecognized effect of progesterone in regulating sperm "behavior" during fertilization.
Highlights
Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes
Exposure of human spermatozoa to a concentration gradient of progesterone (0 –3 M) to simulate approach to the oocyte induced a slowly developing increase in [Ca2؉]i upon which, in many cells, slow oscillations were superimposed. [Ca2؉]i oscillations often started at very low progesterone (
An initial large [Ca2ϩ]i transient, which has been characteristic of all previous reports of the action of progesterone applied as a bolus, was never seen
Summary
Materials—All chemicals were cell culture-tested grade where available. Oregon Green BAPTA-1/AM and BODIPY FL-X ryanodine were from Molecular Probes, Inc. The outflow from the imaging chamber was collected at various time points during a number of the gradient experiments, and progesterone was measured using the ELISA kit following the manufacturer’s instructions. (A control was carried out to confirm that this process did not significantly increase the number of acrosome-reacted cells.) FITC-conjugated P. sativum agglutinin (0.2 mg/ml) was introduced into the chamber and left for 45 min, and distilled water was perfused through the chamber for 10 min. This procedure was carried out at 25 °C.
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