Sánchez Toranzo G, López L A, Martínez J Zapata, Bühler M C Gramajo, Bühler M I
Departamento de Biología del Desarrollo, Instituto Superior de Investigaciones Biológicas (INSIBIO) y Universidad Nacional de Tucumán (UNT), San Miguel de Tucumán, Argentina.
Zygote. 2010 Feb;18(1):41-9. doi: 10.1017/S0967199409990086. Epub 2009 Aug 10.
Dehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts of Artemisia douglasiana Besser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ring. It has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2 of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner in Bufo arenarum fully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown. The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation of Bufo arenarum oocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL). The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL. Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed. Our results indicate that the inhibitory effect on meiosis progression of DhL does not depend only on the activity of the alpha-methylenelactone function, as its hydrogenated derivative, 2H-DhL, in which this function has been inactivated, causes similar effects on amphibian oocytes. However, 2H-DhL was less active than DhL as higher doses were required to obtain a significant inhibition. On the other hand, the analysis of the participation of certain mediators in some of the signalling pathways leading to MPF activation suggests that the Myt1 kinase could be a target of these lactones, while cdc25 phosphatase would not be affected. Besides, the PKA inhibition assays indicate that these lactones would act earlier in the signalling pathways.
脱氢白苞蒿素（DhL）是一种倍半萜内酯，从阿根廷的药用植物道格拉斯蒿（Artemisia douglasiana Besser）的地上部分分离纯化得到。DhL是一个α-亚甲基丁酰-γ-内酯环连接到一个与环外α,β-不饱和环戊烯酮环稠合的七元环。先前已经表明，DhL能选择性地诱导分生组织细胞和血管平滑肌细胞在G2期出现剂量依赖性的短暂停滞。用DhL处理能以剂量依赖性方式抑制处于减数分裂I前期G2期停滞的南美蟾蜍（Bufo arenarum）完全成熟卵母细胞的自发成熟和孕酮诱导的成熟。然而，该过程中涉及的机制的本质仍然未知。这项工作的目的是分析DhL的α-亚甲基-γ-内酯功能是否是其对南美蟾蜍卵母细胞减数分裂重新启动的抑制作用的原因，以及使用α-亚甲基内酯失活的DhL衍生物11,13-二氢-脱氢白苞蒿素（2H-DhL）分析可能参与此效应的一些转导途径。在成熟促进因子（MPF）扩增实验中，通过注射含有活性MPF的细胞质和生发泡内容物来使用2H-DhL，结果与用DhL获得的结果相似，这表明氢化衍生物的作用方式与DhL相似。用DhL或2H-DhL预处理不影响蛋白激酶A（PKA）抑制剂H89诱导的生发泡破裂（GVBD）百分比，这表明这些内酯将作用于诱导MPF激活的信号通路的另一个步骤。DhL和2H-Dhl均抑制微注射冈田酸诱导的GVBD这一事实表明它们可能作用于Myt1激酶的活性。注射生发泡内容物的实验支持了这一观点，在该实验中也观察到了这些内酯对GVBD的抑制作用。我们的结果表明，DhL对减数分裂进程的抑制作用不仅取决于α-亚甲基内酯功能的活性，因为其氢化衍生物2H-DhL（其中该功能已失活）对两栖类卵母细胞产生类似的影响。然而，2H-DhL的活性低于DhL，因为需要更高的剂量才能获得显著的抑制作用。另一方面，对某些介导物参与导致MPF激活的一些信号通路的分析表明，Myt1激酶可能是这些内酯的作用靶点，而细胞周期蛋白依赖性激酶25（cdc25）磷酸酶不会受到影响。此外，PKA抑制试验表明这些内酯将在信号通路中更早发挥作用。
