SPERM, INOSITOL TRISPHOSPHATE, AND THIMEROSAL-INDUCED INTRACELLULAR CA2+ ELEVATIONS IN RABBIT EGGS

Fertilization-Induced calcium (Ca2+) changes were examined in in vivo fertilized rabbit eggs, using the fluorescent Ca2+ indicator fura-2 dextran. Twenty-four of 48 fertilized eggs exhibited repetitive Ca2+ rises with intervals of 13 ± 1 min (means ± SEM) during the recording period of 45 min. None of the unfertilized eggs showed Ca2+ rises (6/6). The mean peak Ca2+ concentration ([Ca2+]1) was 466 ± 30 nM and the average duration was 100 ± 3 sec. The amplitude of the Ca2+ rises decreased and the duration increased as the stage of fertilization progressed from recently fertilized to pronuclear apposition (P < 0.05). In unfertilized eggs, Ca2+ release was elicited by injection of inositol 1,4,6 trisphosphate (InsP3; 5 μM in the injection pipette) with a mean peak [Ca2+]1, of 764 ± 88 nM and a duration of 28 ± 1 sec (n = 9). Injection of InsP3S3 (500 μM), a nonmetabolizable analogue of InsP3, Induced repetitive Ca2+ rises different from sperm-induced rises in periodicity and duration. Exposure to 400 μM thimerosal caused spontaneous Ca2+ rises (1.4 ± 0.1 Ca2+ rises in 45 min of measurements) with an amplitude of 1200 ± 54 nM and duration of 114 ± 8 sec. Heparin injection (100 mg/ml), an InsP3 receptor antagonist, blocked both InsP3 and thimerosal-induced spontaneous Ca2+ rises. Successive application of InsP3 and thimerosal in Ga2+-free medium showed that either InsP3 or thimerosal produced smaller Ca2+ rise(s) when preceded by Ca2+ rise(s) induced by the other agonist. The results of this study indicate that rabbit eggs, like other mammalian eggs, exhibit repetitive Ca2+ rises during fertilization. InsP3 and thimerosal stimulate intracellular Ca2+ release most likely from a common large intracellular pool by activating the InsP3 receptor. © 1993 by Academic Press, Inc.