MECHANISM OF CALCIUM OSCILLATIONS IN FERTILIZED RABBIT EGGS

Changes in intracellular calcium concentrations ([Ca2+](i)) occur at r egular intervals following fertilization in eggs of all mammalian spec ies 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 parti cipate in their generation because injection of heparin (100 mg/ml in the pipette), a competitive InsP3 receptor antagonist, blocked or cons iderably delayed the fertilization [Ca2+](i) rises in all eggs (8/8). Injection of guanosine 5'-0-(2-thiodiphosphate) (GDP beta[S]), a G-pro tein antagonist, which possibly reduced the production of InsP3, also resulted in inhibition of [Ca2+](i) oscillations (n = 7). Ca-2+ inject ion-induced Ca2+ release in fertilized eggs was observed by injection of CaCl2, which evoked intracellular Ca2+ release in all oscillating e ggs (n = 14), but only in a few late fertilization stage nonoscillatin g eggs (7/19 eggs) and in none of the unfertilized eggs (n = 11). Inje ction of InsP3 (5 mu M) between fertilization [Ca2+](i) rises also eli cited Ca2+ responses that were similar in peak [Ca2+](i) to the fertil ization [Ca2+](i) rises (n = 5). In unfertilized eggs, injection of gu anosine 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+ re sponses in 5/7 eggs. The results of this study indicate that IICR part icipates 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 t o reach threshold levels of InsP3 may dictate the periodicity of ferti lization [Ca2+](i) rises in rabbit eggs. (C) 1994 Academic Press, Inc.