The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes
The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes
复制标题
DOI：
10.1016/j.ydbio.2005.08.019
复制
发表时间：
2005-11-15
影响因子：
2.7
通讯作者：
Conti, M
中科院分区：
文献类型：
--
作者：
Hinckley, M;Vaccari, S;Conti, M
在哺乳动物和两栖类卵母细胞中，G2/M期的减数分裂停滞依赖于cAMP的调节。由于在卵母细胞中表达的磷酸二酯酶的遗传失活阻止了重新进入细胞周期，这意味着cAMP的自主合成，因此我们研究了啮齿动物卵母细胞中G蛋白偶联受体(GPCRs)的存在和性质。使用三种独立的策略来确定表达模式，包括GV卵母细胞mRNAs的微阵列分析、EST数据库扫描以及针对GPCR超家族中保守的跨膜区使用简并的引物进行RT-PCR扩增。来自大鼠和小鼠卵母细胞的GPCRmRNAs聚在一起表明，与之密切相关的Gpr3、GPR12和Edg3表达，它们识别鞘氨醇及其代谢产物为配体。用RT-PCR和原位杂交证实了这些mRNAs的表达。这些受体参与了卵母细胞内cAMP水平的控制，这一发现表明，在PDE3A缺乏的卵母细胞中，Gpr3和GPR12的mRNA表达下调，这些卵母细胞的cAMP水平长期升高。在非洲爪哇卵母细胞中表达GPR3或GPR12可阻止孕酮诱导的减数分裂成熟，而表达FSHR则没有影响。在小鼠卵母细胞中注射GPR3或GPR12基因后，卵母细胞的自发成熟也受到抑制。GPR3和GPR12的下调分别导致小鼠和大鼠卵母细胞减数分裂的恢复。然而，去除小鼠的GPR12基因并没有导致泄漏的减数分裂停滞，这表明Gpr3起到了补偿作用。小鼠卵母细胞与GPR3/12配体SPC和S1P的孵育延迟了卵母细胞的自发成熟。我们认为，维持小鼠和大鼠卵母细胞减数分裂停滞所需的cAMP水平取决于GPR3和/或GPR12的表达。(C)2005 Elsevier Inc.保留所有权利。
In mammalian and amphibian oocytes, the meiotic arrest at the G2/M transition is dependent on cAMP regulation. Because genetic inactivation of a phosphodiesterase expressed in oocytes prevents reentry into the cell cycle, suggesting autonomous cAMP synthesis, we investigated the presence and properties of the G-protein-coupled receptors (GPCRs) in rodent oocytes. The pattern of expression was defined using three independent strategies, including microarray analysis of GV oocyte mRNAs, EST database scanning, and RT-PCR amplification with degenerated primers against transmembrane regions conserved in the GPCR superfamily. Clustering of the GPCR mRNAs from rat and mouse oocytes indicated the expression of the closely related Gpr3, Gpr12, and Edg3, which recognize sphingosine and its metabolites as ligands. Expression of these mRNAs was confirmed by RT-PCR with specific primers as well as by in situ hybridization. That these receptors are involved in the control of cAMP levels in oocytes was indicated by the finding that expression of the mRNA for Gpr3 and Gpr12 is downregulated in Pde3a-deficient oocytes, which have a chronic elevation of cAMP levels. Expression of GPR3 or GPR12 in Xenopus laevis oocytes prevented progesterone-induced meiotic maturation, whereas expression of FSHR had no effect. A block in spontaneous oocyte maturation was also induced when Gpr3 or Gpr12 mRNA was injected into mouse oocytes. Downregulation of GPR3 and GPR12 caused meiotic resumption in mouse and rat oocytes, respectively. However, ablation of the Gpr12 gene in the mouse did not cause a leaky meiotic arrest, suggesting compensation by Gpr3. Incubation of mouse oocytes with the GPR3/12 ligands SPC and S1P delayed spontaneous oocyte maturation. We propose that the cAMP levels required for maintaining meiotic arrest in mouse and rat oocytes are dependent on the expression of Gpr3 and/or Gpr12. (C) 2005 Elsevier Inc. All rights reserved.