Studies on the Mechanism for Ca i-Transients in Sea Urchin Zygotes Caused by Refertilization and External Application of Sperm Extract

Abstract

Sea urchin zygotes can be refertilized when they are deprived of the fertilization membrane and the hyaline layer. We have earlier reported that a transient increase of the intracellular Ca 2+concentration (Ca i-transient) is induced in zygotes refertilized by sperm or treated with a sperm extract (spex) (M. Osawa et al.,1994, Dev. Biol.166, 268–276). We investigated quantitative characteristics of the Ca i-transient induced by sperm and spex, using a Ca 2+indicator, Indo-1. When sperm or spex was applied to zygotes, the peak value of the Ca i-transient was 1.16 or 0.69 μ M, respectively. Although these values were lower than the peak value of 1.95 μ Mmeasured during normal fertilization, the entire time courses of the three types of Ca i-transients were similar. The Ca i-transients during fertilization is known to be caused both by the IP 3-induced Ca 2+release (IICR) and by a mechanism independent of IICR. The Ca i-transients during refertilization and fertilization were not inhibited by an IP 3receptor inhibitor, heparin or by a G-protein inhibitor, GDPβS. However, heparin delayed the time courses of both Ca i-transients. These results suggest that there may be two signal transduction pathways operating during refertilization, one dependent and the other independent of IICR. By contrast, both heparin and GDPβS inhibited the spex-induced Ca i-transient. The IP 3content in spex-treated zygotes increased, and the spex-induced Ca i-transient occurred even in the absence of external Ca 2+. Ca i-transient was not observed when spex was injected into zygotes. These data suggest that spex induces IICR in zygotes by activating certain cell surface receptors coupled to G-proteins.