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- Sperm Factor Induces Intracellular Free Calcium: RESULTS(1)

RESULTS(1) U73122 Inhibits SF-Induced [Ca2+]-: Oscillations U73122, a membrane-permeable inhibitor of events leading to PLC activation and/or of PLC itself, was used in this study to evaluate whether SF-induced [Ca2+]i oscillations are mediated by activation of the PI pathway. Incubation of mouse eggs in 20 ^M U73122 for 15 min completely suppressed the ability of SF to initiate [Ca2+]i oscillations (n = 0/7; Fig. 1B). Incubation of eggs with lower concentrations of the inhibitor was not as effective, and SF-induced Ca2+ responses were blocked in only 1/5 (10 ^M) and 7/14 (15 ^M) eggs (Fig. 1E), although in the few eggs in which the responses were inhibited the inhibition was complete.

Incubation with higher U73122 concentrations (25 ^M), although effective in blocking Ca2+ responses, resulted in cell death within a couple of hours after exposure, and this higher concentration was not used in our studies. Injection of SF induced normal [Ca2+] oscillations in untreated eggs (n = 8/8; Fig. 1A) or into eggs incubated for 15 min in 20 ^M U73343 (n = 4/4), the inactive analogue of U73122 (Fig. 1C; P < 0.05). In spite of successful inhibition of SF-induced Ca2+ responses by U73122, several detrimental effects have been reported in cells exposed to this inhibitor, including a rise in basal [Ca2+]i levels and emptying of IP3-sensitive internal Ca2+ stores. Thus, to determine if the inhibition of the Ca2+ responses in our studies was due to emptying of the Ca2+ stores, eggs incubated with 20 ^M U73122 were injected with 500 ^M IP3 (concentration in the pipette). This concentration of IP3 was chosen because it closely reproduced in frequency and duration the oscillations evoked by injection of 0.5 mg/ml SF. Injection of IP3, which acts directly on the IP3R in the ER, induced [Ca2+]i oscillations in all eggs (Fig. 1D; n = 4/4). These results suggest that the inhibitory effect of U73122 on SF-induced [Ca2+]i oscillations is most likely due to its ability to prevent activation of the PI pathway and subsequent IP3 production. Fig1Sperm Factor Induces Intracellular FIG. 1. Incubation of mouse eggs in U73122 inhibits SF-induced [Ca2+] oscillations. A) Control eggs show normal [Ca2+]j oscillations upon SF injection. B) Incubation of eggs in U73122 (20 ц,М) inhibits SF-induced [Ca2+] oscillations. C) Incubation in U73343 (20 ц,М) fails to inhibit [Ca2+] oscillations. D) U73122 (20 ц,М) did not inhibit [Ca2+] oscillations induced by injection of IP3 (500 ц,М). SF concentration was 0.5 mg/ml in the injection pipette throughout these studies, resulting in intracellular SF amounts representing 1-2.5 sperm equivalents. Arrows indicate times of SF or IP3 injection, respectively. The pipette was removed immediately after the injection in all cases. E) Percentage inhibition of [Ca2+] oscillations when eggs are incubated in various concentrations of U73122. Error bars represent SEM. Different superscript letters denote significant differences (P < 0.05; chi-square test).

September 23, 2013 Sperm
Tags: calcium developmental biology fertilization