Localization of inositol 1,4,5 trisphosphate receptor type1 (IP_3R1), the channel responsible for Ca^<2+> release and oscillations during fertilization in mammals

在哺乳动物中，精子诱导的细胞内Ca^<2+>信号（[Ca^<2+>]i）在受精过程中介导减数分裂的退出和卵母细胞的激活。最近，研究表明，在小鼠卵母细胞中，肌醇1,4,5三磷酸受体1 （IP_3R1）的磷酸化水平在成熟和受精过程中发生了变化（Ito et al., 2008, Dev. Biol）， IP_3R1是受精过程中负责Ca^<2+>释放和振荡的通道。然而，IP3R1在其他物种，特别是卵母细胞发育能力较低的家养物种中应用胞浆内单精子注射（ICSI）和体细胞核移植（SCNT）时的动力学尚不清楚。因此，我们检测了IP_3R1在猪卵母细胞体外成熟过程中的表达、磷酸化和定位。在这里，我们目前的研究表明，IP_3R1蛋白的表达在成熟过程中没有改变，尽管受体的磷酸化状态在生发囊泡分解（GVBD）之后显着上调。在GV期，IP_3R1遍布细胞质。卵母细胞成熟后，证实了卵母细胞皮层IP_3R1蛋白的浓度。在成熟卵母细胞中，大簇IP_3R1在卵母细胞皮层中形成，除了在靠近纺锤体的皮层环状带中形成。综上所述，我们的研究结果表明，IP_3R1在成熟过程中经历了动态磷酸化和定位，这可能是受精时振荡产生的基础。我们的发现将有助于提高国内物种的ICSI和SCNT的效率。

In mammals, a sperm-induced intracellular Ca^<2+> signal ([Ca^<2+>]i) mediates both exit of meiosis and oocyte activation during fertilization. Recently, it was demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP_3R1), the channel responsible for Ca^<2+> release and oscillations during fertilization, changed during maturation and fertilization (Ito et al., 2008, Dev. Biol). However, kinetics of IP3R1 is not well understood in other species, especially domestic species which have low developmental ability of oocytes when intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) are applied. Therefore, we examined the expression, phosphorylation and localization of IP_3R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP_3R1 protein did not change during maturation, although the phosphorylation status of the receptor was dramatically upregulated beyond germinal vesicle break down (GVBD). At the GV stage, IP_3R1 was observed throughout the cytoplasm. Following oocyte maturation, concentration of IP_3R1 protein at the cortex of the oocyte was confirmed. In matured oocytes, large clusters of IP_3R1 were formed in the cortex of the oocyte except in a ring-shaped band of cortex adjacent to the spindle. Taken together, our results show that IP_3R1 undergoes dynamic phosphorylation and localization during maturation and this might underlie the generation of oscillations at fertilization. Our findings will contribute to improve the efficiency of ICSI and SCNT in domestic species.