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+ inducer fura-2 dextran. Twenty-four of 48 fertilized eggs exhibited repetitive Ca2+ rises with intervals of 13 +/- 1 min (mean +/- SEM) during the recording period of 45 min. None of the unfertilized eggs showed Ca2+ rises (6/6). The mean peak Ca2+ concentration ([Ca2+]i) 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 1,4,5 trisphosphate (InsP3; 5 microM in the injection pipette) with a mean peak [Ca2+]i of 764 +/- 88 nM and a duration of 28 +/- 1 sec (n = 9). Injection of InsP3S3 (500 microM), a nonmetabolizable analogue of InsP3, induced repetitive Ca2+ rises different from sperm-induced rises in periodicity and duration. Exposure to 400 microM 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 Ca(2+)-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.