Nuccitelli R, Yim D L, Smart T
Section of Molecular and Cellular Biology, Division of Biological Sciences, University of California, Davis 95616.
Dev Biol. 1993 Jul;158(1):200-12. doi: 10.1006/dbio.1993.1179.
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)
我们利用非洲爪蟾卵母细胞中fura - 2的荧光比率成像技术，研究受精时通常触发的游离Ca2 +增加波的起始和传播。将天然成熟的带卵胶膜的卵母细胞注射fura - 2，并在添加精子时用350和385 nm的荧光激发进行比率成像。[Ca2 +]i升高通常始于卵表面附近的一个小点，在20 - 60秒内保持相当局限，然后以7.5±0.05微米/秒的速度更快地扩散到整个卵，在受精后约5分钟到达对侧。通过将细胞质中fura - 2的浓度增加到100微摩尔/升，[Ca2 +]i波的速度减慢，而250微摩尔/升的fura - 2则阻止波的传播。精子激活波中的[Ca2 +]i峰值为2.2±0.1微摩尔/升，受精后21±0.7分钟内[Ca2 +]i恢复到激活前水平。我们通过注射干扰Ins(1,4,5)P3诱导的Ca2 +释放或Ca(2 +)诱导的Ca2 +释放（CICR）的物质，进一步研究了精子触发Ca2 +波的机制。肝素（3 kDa）以与其细胞质浓度成线性比例的方式抑制精子诱导的卵母细胞激活。在13微摩尔/升（390微克/毫升）时，精子诱导的激活被完全阻断，在7.5微摩尔/升（225微克/毫升）时，一半卵母细胞的激活被抑制。使用Hoechst染料标记核DNA证实，所有注射13微摩尔/升肝素的卵都是多精受精的。对注射7.5微摩尔/升肝素的卵进行成像显示，[Ca2 +]i增加的多个短暂“斑点”未能扩散到整个卵。注射针对PIP2的单克隆抗体（0.2微摩尔/升）kt3g，在注射的30个卵中有73%阻断了精子诱导的卵母细胞激活，这表明激活需要卵膜中PIP2的水解，而不仅仅是精子引入Ins(1,4,5)P3。这种精子诱导的卵母细胞激活不受两种CICR抑制剂普鲁卡因（mM）或钌红（30微摩尔/升）的阻断，并且两种CICR刺激剂咖啡因（10 mM）或ryanodine（50微摩尔/升）均未触发卵母细胞激活。（摘要截短于250字）
