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 (IP3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca2+ and released it in response to IP3 in a concentration-dependent manner. Repeated additions of IP3 were unable to cause further release. Heparin inhibited Ca2+ release in response to IP3. The homogenates also released Ca2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP3-gated Ca2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca2+. At low concentrations, Tg caused a slow release of Ca2+; at higher concentrations, it elicited a rapid release. Release of Ca2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca2+ release in Chaetopterus egg homogenates. There was no Ca2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP3. Thus, Ca2+ release at fertilization in these oocytes occurs through IP3-gated channels.