The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins(1, 4, 5)P3.
We used fluorescence ratio imaging of fura-2 in the egg of Xenopus laevis to study the initiation and propagation of the wave of increased free Ca2+ that is normally triggered at fertilization. Naturally matured, jellied eggs were injected with fura-2 and ratio-imaged with fluorescence excitation at 350 and 385 nm while sperm were added. The [Ca2+]i rise normally begins as a small spot near the surface of the egg, remains fairly localized for 20-60 sec, and then spreads more rapidly across the egg at 7.5 +/- 0.05 microns/sec to reach the antipode about 5 min after fertilization. The [Ca2+]i wave velocity is slowed by increasing the concentration of fura-2 in the cytoplasm to 100 microM, and 250 microM fura-2 blocks wave propagation. The peak [Ca2+]i in a sperm-activated wave is 2.2 +/- 0.1 microM and [Ca2+]i returns to preactivation levels within 21 +/- 0.7 min after fertilization. We further studied the mechanism by which sperm trigger the Ca2+ wave by injecting substances that interfere with Ins(1,4,5)P3-induced Ca2+ release or Ca(2+)-induced Ca2+ release (CICR). Heparin (3 kDa) inhibits sperm-induced egg activation in a manner that is linearly proportional to its cytoplasmic concentration. At 130 microM (390 micrograms/ml), sperm-induced activation is completely blocked and at 75 microM (225 micrograms/ml) activation of half of the eggs is inhibited. All eggs injected with 130 microM heparin are polyspermic as verified using Hoechst dye to label nuclear DNA. Imaging eggs injected with 75 microM heparin revealed multiple, transient "spots" of increased [Ca2+]i that failed to spread across the egg. Injection of a monoclonal antibody to PIP2 (0.2 microM), kt3g, blocked sperm-induced egg activation in 73% of the 30 eggs injected, suggesting that activation requires the hydrolysis of PIP2 in the membranes of the egg rather than simply the introduction of Ins(1,4,5)P3 from the sperm. This sperm-induced egg activation is not blocked by either of two CICR inhibitors, procaine (10 mM) or ruthenium red (30 microM), and egg activation is not triggered by either of two stimulators of CICR, caffeine (10 mM) or ryanodine (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Nuccitelli
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Section of Molecular and Cellular Biology, Division of Biological Sciences, University of California, Davis 95616.
Yim
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Smart
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D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008297:Male; D011343:Procaine / Q000494:pharmacology; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D014982:Xenopus laevis / Q000196:embryology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D012430:Ruthenium Red; C053459:5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D012433:Ryanodine; D002110:Caffeine; D011343:Procaine; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D002118:Calcium