Mechanism of calcium oscillations in fertilized rabbit eggs.
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) (GDP beta[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 microM) 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.
Fissore
R A
RA
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst 01003.
Robl
J M
JM
D000818:Animals; D002118:Calcium / Q000502:physiology*; D005260:Female; D005306:Fertilization; D019204:GTP-Binding Proteins / Q000502:physiology; D006150:Guanine Nucleotides / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D008297:Male; D008845:Microinjections; D010063:Ovum / Q000502:physiology*; D011817:Rabbits
D006150:Guanine Nucleotides; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D019204:GTP-Binding Proteins; D002118:Calcium