Mechanisms of calcium release and sequestration in eggs of Chaetopterus pergamentaceus

Abstract

Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP 3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca 2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca 2+ and released it in response to IP 3 in a concentration-dependent manner. Repeated additions of IP 3 were unable to cause further release. Heparin inhibited Ca 2+ release in response to IP 3. The homogenates also released Ca 2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP 3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP 3-gated Ca 2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca 2+. At low concentrations, Tg caused a slow release of Ca 2+; at higher concentrations, it elicited a rapid release. Release of Ca 2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca 2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca 2+ release in Chaetopterus egg homogenates. There was no Ca 2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP 3. Thus, Ca 2+ release at fertilization in these oocytes occurs through IP 3-gated channels.