Cell Reproduction Direct Evidence for Ionic Messengers in the Two Phases of Metabolic Derepression at Fertilization in the Sea Urchin Egg

It has been proposed that the release of intracellular ionic calcium may be the universal factor promoting activation of egg metabolism at fertilization. If this hypothesis is valid, then fertilization and all parthenogenetic agents which evoke the entire program of activation would necessarily involve an intracellular rise in ionic calcium. Fertilization or parthenogenetic activation of Lytechinus pictus eggs was monitored after injection with the Ca-sensitive photoprotein aequorin. We estimated the peak calcium transient at 2.5-4.5 jiM free calcium, 45-60 s after activation and lasting 2-3 mm, assuming equal distribution throughout the cytoplasm. The calcium release at fertilization was shown to be from intracellular stores.The threshold for the discharge of the cortical vesicles was between 9-18 pM calcium, suggesting that the transient calcium release is confined to the inner subsurface of the egg. Parthenogenetic treatments, ionophore A23187, non-electrolyte 1M urea and hypertonic medium 1.6 x concentrated sea water, all acted to release calcium from intracellular stores. Sperm, ionophore and non-electrolyte release from the same calcium store which can be recharged after 40 mm. Hypertonic medium releases from a different store. It has been proposed that the second ionic messenger is an increase in intracellular pH, which is responsible for the late steps in activation such as protein and DNA syntheses. For this hypothesis to be valid, treatment with weak bases, which only evoke late steps in activation, should not release intracellular calcium but should raise intracellular pH. Measurements with aequorin injected eggs showed some calcium entry from external solutions with NH4C1 exposures but that it was not essential to weak base activation. Measurement with microelectrodes of intracellular pH in intact eggs did show a rise in pH with NH4C1 and other weak bases as well as in normal fertilization. 1This work was supported by NSF PCM 74-0943OA01 and 7704260 to R.A.S., and BNS 75-20288 to R.S.Z. 415 Copyrighs3 1978 by Academic Press, Inc. All right of reproduction in any form reserved.