Hinckley Mary, Vaccari Sergio, Horner Kathleen, Chen Ruby, Conti Marco
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5317, USA.
Dev Biol. 2005 Nov 15;287(2):249-61. doi: 10.1016/j.ydbio.2005.08.019. Epub 2005 Oct 17.
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.
在哺乳动物和两栖动物的卵母细胞中，减数分裂在G2/M转换期的停滞依赖于cAMP调节。由于卵母细胞中表达的一种磷酸二酯酶的基因失活会阻止细胞重新进入细胞周期，这表明存在自主cAMP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）的存在及其特性。使用三种独立策略确定了表达模式，包括对生发泡（GV）期卵母细胞mRNA进行微阵列分析、扫描EST数据库以及用针对GPCR超家族中保守跨膜区域的简并引物进行RT-PCR扩增。对大鼠和小鼠卵母细胞的GPCR mRNA进行聚类分析表明，密切相关的Gpr3、Gpr12和Edg3表达，它们识别鞘氨醇及其代谢产物作为配体。通过用特异性引物进行RT-PCR以及原位杂交证实了这些mRNA的表达。Gpr3和Gpr12的mRNA在Pde3a缺陷型卵母细胞中表达下调，而这些卵母细胞的cAMP水平长期升高，这一发现表明这些受体参与了卵母细胞中cAMP水平的控制。在非洲爪蟾卵母细胞中表达GPR3或GPR12可阻止孕酮诱导的减数分裂成熟，而表达促卵泡激素受体（FSHR）则没有影响。当将Gpr3或Gpr12 mRNA注射到小鼠卵母细胞中时，也会诱导自发卵母细胞成熟受阻。GPR3和GPR12的下调分别导致小鼠和大鼠卵母细胞减数分裂恢复。然而，在小鼠中敲除Gpr12基因并未导致减数分裂停滞出现渗漏，这表明可能由Gpr3进行补偿。用GPR3/12配体鞘氨醇磷酸胆碱（SPC）和1-磷酸鞘氨醇（S1P）孵育小鼠卵母细胞会延迟自发卵母细胞成熟。我们提出，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。
