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Abstract：
We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca2+ ([Ca2+]i): transient elevations in [Ca2+]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca2+ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca2+]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca2+ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca2+]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca2+ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca2+]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca2+ wave and spiral-like Ca2+ oscillations, respectively.
摘要：
我们以前报道过，日本鹌鹑的卵激活是由两种不同类型的细胞内Ca2（[Ca2]i）驱动的：由磷脂酶Czeta1（PLCZ1）诱导的[Ca2]i瞬时升高和持续的螺旋状Ca2振荡通过柠檬酸合酶（CS）和aconitate水合酶2（ACO2）。尽管在显微注射PLCZ1，CS之前阻断了肌醇1,4,5-三磷酸受体（ITPR），和ACO2cRNAs仅阻止[Ca2+]i的瞬时增加，微量注射ryanodine受体激动剂（RYRs）引起的螺旋状Ca2振荡，表明ITPR和RYR都参与了这些事件。在这项研究中,我们调查了ITPRs和RYRs的同种型，这些同种型负责两种类型的[Ca2]i的表达增加。RT-PCR和蛋白质印迹分析显示ITPR1、ITPR3和RYR3在排卵卵中表达。这些蛋白在PLCZ1、CS、和ACO2cRNAs，这是卵激活完成的时间。然而,在单次注射PLCZ1cRNA后30分钟开始降解ITPR1和ITPR3，而不是RYR3，对应于初始Ca2+波终止的时间。相比之下,在显微注射CS和ACO2cRNA后3小时观察到RYR3降解。这些结果表明，在日本鹌鹑的卵激活过程中，ITPR和RYR3差异介导[Ca2]i的折痕，ITPR和RYR3介导的事件的下调终止了最初的Ca2+波和螺旋状Ca2+振荡，分别。
