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，精子诱导的细胞内钙信号([Ca^&lt；2+&gt；]i)在受精过程中既介导了减数分裂的退出，也介导了卵母细胞的激活。最近，在小鼠卵母细胞中发现，在成熟和受精过程中，肌醇1，4，5三磷酸受体1(IP_3R1)的磷酸化水平发生了变化(Ito等，2008，Dev.)Biol)。然而，在其他物种，特别是在卵胞浆内单精子注射(ICSI)和体细胞核移植(SCNT)时卵母细胞发育能力较低的家养物种中，IP3R1的动力学还不是很清楚。因此，我们检测了IP3R1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，我们的研究表明，IP_3R1蛋白的表达在成熟过程中没有变化，尽管在生发泡破裂(GVBD)后，该受体的磷酸化状态显著上调。在GV期，IP_3R1分布于整个细胞质。卵母细胞成熟后，卵母细胞皮质中可见IP_3R1蛋白的表达。在成熟卵母细胞中，除了纺锤体附近的环状皮质带外，在卵母细胞的皮质中形成了大的IP_3R1簇。综上所述，我们的结果表明，IP_3R1在成熟过程中经历了动态的磷酸化和定位，这可能是受精时产生振荡的基础。我们的发现将有助于提高ICSI和SCNT在家养物种中的效率。

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.