To assess whether a component(s) of SF was the target of the inhibitory action of U73122, SF was incubated with the inhibitor prior to injection into eggs. Injection of SF preincubated with 30 ^M U73122 abrogated the ability of SF to initiate [Ca2+]i oscillations (n = 5/19; Fig. 2B), whereas injection of untreated SF (n = 4/4; Fig. 2A) or injection of SF preincubated with 30 ^M U73343 (n = 4/4; Fig. 2C) induced normal Ca2+ responses (P < 0.05). Incubation of SF with lower concentrations of the inhibitor was much less effective; 0/3 (10 ^M) and 4/9 (20 ^M) SF-injected eggs showed inhibition of oscillations (Fig. 2H). In these experiments, the mixture of SF + 30 ^M U73122 was injected into the eggs and it is possible that the injected inhibitor could be acting on an egg molecule rather than inhibiting a sperm component.
To test this possibility, 30 ^M U73122 was injected into eggs (approximate intracellular concentration of 0.5-1 ^M), followed within 10 min by injection of SF. All eggs treated in this manner exhibited [Ca2+]i oscillations (n = 4/4; Fig. 2D), indicating that U73122 was most likely inhibiting a molecule in SF. In addition, we also determined that Injection of 30 ^M U73122 alone did not cause Ca2+ release or an increase in the egg’s basal [Ca2+]i levels (n = 4/4; Fig. 2E), suggesting that the concentration of the inhibitor used in our studies was not toxic to eggs.
The inhibitory effects of U73122, whose active site contains an electrophilic maleimide group, have been reported to be lost when exposed to reducing agents. To test whether the effects of U73122 on SF activity were reversible by the same mechanism, SF was preincubated simultaneously for 15 min with 30 ^M U73122 and 10 mM DTT and then injected into eggs or SF and U73122 were preincubated first and 15 min later DTT was added to the incubation mixture. In both situations, exposure to DTT reversed the inhibitory effects of U73122 on SF (n = 3/3, Fig. 2F; n = 4/4, Fig. 2G; respectively).
FIG. 2. Incubation of SF with U73122 inhibits its ability to initiate [Ca2+] oscillations, and this effect is reversed by DTT. A) Control eggs show normal [Ca2+] oscillations when injected with untreated SF. B) When SF is incubated in U73122 (30 ^M), its ability to cause [Ca2+] oscillations is suppressed (C), but incubation of SF in U73343 (30 ^M) does not inhibit [Ca2+] responses by SF. D) Inhibition appears specific to an SF component; prior injection of eggs with U73122 (30 ^M) fails to block induction of Ca2+ responses by SF. E) Injection of U73122 (30 ^M) alone does not cause [Ca2+]| release. DTT (10 mM) reversed the effect of U73122 on SF whether it was incubated simultaneously with SF + U73122 (F) or added to the U73122 + SF mixture after a 15-min preincubation (G). H) Percentage inhibition of [Ca2+]| oscillations when SF is incubated with various concentrations of U73122. Error bars represent SEM. Different superscript letters denote significant differences (P < 0.05; chi-square test).