Orai1 mediates store-operated Ca²⁺ entry during fertilization in mammalian oocytes

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• 作者：Chunmin Wang^a ; Kiho Lee^b ; Erzsé ; bet Gajdó ; csi^c ; Á ; gnes Bali Papp^c ; Zoltan Machaty^a ; ^{zmachaty@purdue.edu}
• 关键词：Calcium ; Signaling ; Oocyte ; Fertilization ; Orai1 ; Calcium channel
• 刊名：Developmental Biology
• 出版年：2012
• 期刊代码：167_00121606
• 类别：bio
• 出版时间：15 May, 2012
• 卷：365
• 期：2
• 页码：414-423
• 文件大小：1025 K

摘要

The presence of the store-operated Ca²⁺ entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921 bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca²⁺ influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca²⁺ influx after store depletion and subsequent Ca²⁺ add-back; the Ca²⁺ oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca²⁺ entry and had an inhibitory effect on the fertilization Ca²⁺ signal. The abnormal Ca²⁺ signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca²⁺ entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca²⁺ channel that is responsible for mediating Ca²⁺ entry after the mobilization of intracellular Ca²⁺ in oocytes. Orai1 and a functional store-operated Ca²⁺ entry pathway are required to maintain the Ca²⁺ oscillations at fertilization and to support proper embryo development.