Hoang Yvonne D, Avakian Ani P, Luderer Ulrike
Division of Occupational and Environmental Medicine, Department of Medicine, University of California at Irvine, 5201 California Avenue, Suite 100, 92617, USA.
Reprod Toxicol. 2006 Feb;21(2):186-96. doi: 10.1016/j.reprotox.2005.07.011. Epub 2005 Sep 23.
The antioxidant tripeptide glutathione (GSH) protects ovarian follicles against oxidative damage that may lead to apoptotic death. The rate-limiting step in synthesis of GSH is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic subunit (GCLC), and a modifier subunit (GCLM). We hypothesized that GSH depletion in vivo or in vitro with buthionine sulfoximine (BSO), a specific inhibitor of GCL activity, would increase ovarian and granulosa cell GCL subunit expression. Ovarian glutathione levels are lowest on proestrous morning and increase to their highest levels on estrus and metestrus. Therefore, we treated rats on proestrous morning or on proestrous morning and again 12h later to prevent the normal increase in ovarian glutathione between proestrus and estrus. Ovarian Gclc and Gclm mRNA levels and GCLC protein levels increased transiently by 1.4-1.5-fold at 8 h, but not at 12 or 24 h, after a single dose of BSO administered to adult rats on the morning of proestrus. GCLC protein levels were also modestly increased 1.4-fold at 12 h after a second dose of BSO. GCLM protein levels increased 1.4-fold at 24 h after a single dose of BSO, but not at other time points. BSO treatment did not significantly alter ovarian GCL enzymatic activity or the intraovarian localization of either GCL subunit mRNA. Treatment of a human granulosa cell line or primary rat granulosa cells with BSO suppressed intracellular GSH; however, there was no compensatory upregulation of GCL subunit protein or mRNA levels. These results demonstrate that ovarian follicles and granulosa cells are minimally able to respond to acute GSH depletion by upregulating expression of GCL.
抗氧化三肽谷胱甘肽（GSH）可保护卵巢卵泡免受可能导致凋亡性死亡的氧化损伤。GSH合成的限速步骤由谷氨酸半胱氨酸连接酶（GCL）催化，GCL是一种异二聚体，由催化亚基（GCLC）和调节亚基（GCLM）组成。我们推测，用GCL活性的特异性抑制剂丁硫氨酸亚砜胺（BSO）在体内或体外消耗GSH会增加卵巢和颗粒细胞GCL亚基的表达。卵巢谷胱甘肽水平在动情前期早晨最低，在发情期和间情期升至最高水平。因此，我们在动情前期早晨或在动情前期早晨给药并在12小时后再次给药，以阻止卵巢谷胱甘肽在动情前期和发情期之间正常增加。在动情前期早晨给成年大鼠单次注射BSO后，卵巢Gclc和Gclm mRNA水平以及GCLC蛋白水平在8小时时短暂增加1.4 - 1.5倍，但在12或24小时时未增加。在第二次注射BSO后12小时，GCLC蛋白水平也适度增加1.4倍。单次注射BSO后24小时，GCLM蛋白水平增加1.4倍，但在其他时间点未增加。BSO处理未显著改变卵巢GCL酶活性或任一GCL亚基mRNA的卵巢内定位。用BSO处理人颗粒细胞系或原代大鼠颗粒细胞可抑制细胞内GSH；然而，GCL亚基蛋白或mRNA水平没有代偿性上调。这些结果表明，卵巢卵泡和颗粒细胞通过上调GCL表达对急性GSH消耗的反应能力极低。
