Mechanism of Calcium Oscillations in Fertilized Rabbit Eggs

Abstract

Changes in intracellular calcium concentrations ([Ca2+]i) occur at regular intervals following fertilization in eggs of all mammalian species studied to date. To investigate the mechanisms of their generation, rabbit eggs were injected with the fluorescent Ca2+ indicator fura-2 dextran. [Ca2+]i oscillations were associated with fertilization (n = 10) and inositol 1,4,5-trisphosphate (InsP3; IICR) appears to participate in their generation because injection of heparin (100 mg/ml in the pipette), a competitive InsP3 receptor antagonist, blocked or considerably delayed the fertilization [Ca2+]i rises in all eggs (8/8). Injection of guanosine 5′-0-(2-thiodiphosphate) (GDPβ[S]), a G-protein antagonist, which possibly reduced the production of InsP3, also resulted in inhibition of [Ca2+]i oscillations (n = 7). Ca2+ injection-induced Ca2+ release in fertilized eggs was observed by injection of CaCl2, which evoked intracellular Ca2+ release in all oscillating eggs (n = 14), but only in a few late fertilization stage nonoscillating eggs (7/19 eggs) and in none of the unfertilized eggs (n = 11). Injection of InsP3 (5 μM) between fertilization [Ca2+]i rises also elicited Ca2+ responses that were similar in peak [Ca2+]i to the fertilization [Ca2+]i rises (n = 5). In unfertilized eggs, injection of guanosine 5′-0-(3-thiotriphosphate) (GTP[S]; 5-20 mM), a stimulator of G-proteins, induced [Ca2+]i oscillations. CaCl2 injections, delivered between GTP[S]-induced [Ca2+]i rises, resulted in increased Ca2+ responses in 5/7 eggs. The results of this study indicate that IICR participates in the generation of fertilization-associated [Ca2+]i rises and that Ca2+ injection-induced Ca2+ release appears to be stimulated by a product of the phosphoinositide pathway. Furthermore, the time to reach threshold levels of InsP3 may dictate the periodicity of fertilization [Ca2+]i rises in rabbit eggs.