Thapsigargin elevates and potentiates the ability of progesterone to increase intracellular free calcium in human sperm: possible role of perinuclear calcium.

The effect of thapsigargin on intracellular free calcium ([Ca2+]i) was examined in swim up human sperm. Thapsigargin elevated [Ca2+]i in a dose dependent manner, with maximal effects observed with 10 microM. The increase in [Ca2+]i was relatively slow in taking 3-4 min to reach a maximal level. The increase in [Ca2+]i was inhibited when extracellular Ca2+ was chelated with EGTA, thus the source of Ca2+ for the increase was extracellular. The effect of thapsigargin to stimulate Ca2+ influx was also observed using the Mn2+ quenching of intracellular Fura-2. Thapsigargin pre-treatment of sperm was able to potentiate the effects of progesterone to elevate [Ca2+]i. Progesterone pre-treatment also potentiated the ability of thapsigargin to elevate [Ca2+]i. Further evidence to support the idea that thapsigargin was promoting Ca2+ influx was the fact that thapsigargin potentiated ionomycin (an agent that can increase Ca2+ cycling across phospholipid membranes) induced elevations in [Ca2+]i. Conversely, ionomycin also potentiated thapsigargin induced elevations in [Ca2+]i. It is concluded from these studies that high concentrations of thapsigargin are able to stimulate Ca2+ influx in human sperm by a mechanism not involving the endoplasmic reticulum Ca(2+)-ATPase pump since this organelle is absent in mature sperm. However, a likely site of action of thapsigargin in mature sperm is at the level of the nuclear membrane Ca(2+)-ATPase pump which is identical to the endoplasmic reticulum Ca(2+)-ATPase pump and is sensitive to inhibition by thapsigargin.