受精和人工激活诱导的小鼠卵内游离钙离子变化(英文)

Intracellular Ca^(2+) during fertilization and artificial activation in mouse oocytes

目的:研究哺乳类动物卵母细胞激活机制。方法:将休止于第二次成熟分裂中期的小鼠卵母细胞负载Fura 2-AM后用乙醇,钙离子载体,电脉冲或受精等激活;采用Spex AR-CM-MIC阳离子检测系统测定卵激活过程中细胞内游离Ca^(2+)浓度变化;电镜下检测卵激活后皮质颗粒释放。结果:在含Ca^(2+)(1.7mmol·L^(-1))的IVF液中,精子受精诱发卵内Ca^(2+)浓度波动,这种Ca^(2+)波动持续达3—4h直至受精卵发育至原核期消失。当外Ca^(2+)升高至5.0mmol·L^(-1)时Ca^(2+)波动加快;去除外Ca^(2+)后Ca^(2+)波动很快消失;将卵移入含Ca^(2+)IVF后又恢复Ca^(2+)波动。出现Ca^(2+)波动的卵随后均排出第二极体并形成原核。胞内注射Ca^(2+)螯合剂依他酸(200μmol·L^(-1))抑制受精诱导的Ca^(2+)波动则阻止卵激活。胞内注射肝素(MW=3500,100μmol·L^(-1))不能阻止受精Ca^(2+)波动。乙醇,钙离子载体和1次电脉冲刺激诱导卵内Ca^(2+)浓度升高1次并且仅诱导大龄卵(>18h CG)激活。刚排出卵的激活则需要[Ca^(2+)]_i多次升高诱导。人工激活诱导卵出现与受精相似的皮质颗粒释放。卵内注射Ca^(2+)螯合剂抑制[Ca^(2+)]_i升高则阻断卵激活的一系列反应。结论:细胞内游离Ca^(2+)升高是诱导卵激活的重要信号;受精与人工激活诱导的[Ca^(2+)]_i升高及Ca~(

AIM: TO study the mechanism of oocyte activation in mammals. METHODS: Mouse oocytes arrested at metaphase of the second meiotic division were loaded with Fura 2-AM and then activated with ethanol, calcimycin, electric pulse or fertilization. Intracellular free Ca2+ during activation were measured by Spex AR-CM-MIC cation system. Cortical granule exocytosis after activation was detected under electron microscopy. RESULTS: Sperm penetration initiated a long lasting Ca2+ oscillation in Ca2+ containing IVF medium in mouse ova. The Ca2+ oscillation lasted for over 3 - 4 h until the ova developed to pronuclear stage. The Ca2+ oscillated faster as extracellular Ca2+ concentration was increased from normal 1.7 mmol·L-1 to 5.0 mmol·L-1 and ceased to oscillate when extracellular Ca2+ was removed. The ova resumed Ca2+ oscillation after transferred back to IVF (Ca2+ 1.7 mmol·L-1). The ova which exhibited Ca2+ oscillation all extruded second polar body and formed pronuclei. Suppression the Ca2+ oscillation by intracellular injection of egtazic acid (2 - 10 pL, 200 μmol · L-1) blocked the activation of oocytes. Heparin (100 μmol · L-1) injection failed to prevent the Ca2+ oscillation. In artificial activation, ethanol, calcimycin, and a single electric pulse usually induced a monotonic Ca2+ rise and resulted in the activation only in older oocytes ( > 18 h after CG injection). Activation of freshly ovulated oocytes required multiple intracellular Ca2+ increases induced by repetitive electric pulses. Artificial activation elicited the similar cortical granule exocytosis in oocytes as occuring at fertilization. Suppression of the intracellular Ca2+ elevation by introduction of egtazic acid into the oocytes blocked the activation process. CONCLUSIONS: The increase of intracellular free Ca2+ is the primary signal responsible for the initiation of oocyte activation but there are distinct differences between fertilization and artificial activation both in Ca2+ change patterns and Ca2+ sources. Young oocytes require oscillatory Ca2+ signals for activation.