Billig H, Furuta I, Hsueh A J
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317.
Endocrinology. 1994 Jan;134(1):245-52. doi: 10.1210/endo.134.1.8275940.
The majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death. Although GnRH and its agonists have been shown to suppress ovarian growth and differentiation in hypophysectomized rats, studies on the induction of follicle atresia by GnRH are contradictory. In the present study, the direct effect of GnRH on the occurrence of apoptosis in the ovary was investigated in hypophysectomized estrogen-treated immature rats. Starting 2 days after operation and estrogen capsule implantation, rats were treated with a GnRH agonist (GnRHa; [desGly10,D-Phe6,Pro9-N-ethylamide] GnRH; 50 micrograms/injection, twice daily). Total ovarian DNA was isolated 48 h later, labeled at the 3'ends with [32P]dideoxy ATP, and size-fractionated. Compared to that in control animals, treatment with GnRHa increased DNA fragmentation in multiples of 180 basepairs, a hallmark of apoptosis, demonstrating that GnRH directly increases ovarian apoptotic cell demise. In contrast, FSH treatment (10 micrograms/injection, twice daily) decreased apoptotic DNA fragmentation, and the antiapoptotic effect of FSH was partially blocked by concomitant treatment with GnRHa. The apoptosis-inducing effect of GnRHa was time and dose dependent, with a significant increase seen after 24 h of treatment and a maximal 5.5-fold increase with 10 micrograms GnRHa/injection. Similar to studies using DNA isolated from whole ovaries, DNA obtained from isolated granulosa cells also showed a time- and dose-dependent increase in DNA fragmentation after GnRHa treatment. The effect on DNA fragmentation by GnRHa was mediated by ovarian GnRH receptors, because a potent GnRH receptor blocker, Azaline B, prevented GnRHa action. In addition, in situ end labeling of DNA using digoxigenin-dideoxy-UTP showed that DNA fragmentation was confined to the granulosa cells of preantral and antral follicles. No GnRHa-induced apoptosis was detected in granulosa cells of primary follicles or in thecal and interstitial cells. These data suggest that GnRH directly increases apoptotic cell death in the ovary, and the GnRH action is confined to the granulosa cells. These data provide a basis for future studies on the mechanism of follicular atresia and the regulation of ovarian endonuclease by GnRH.
大多数卵巢卵泡通过涉及凋亡性细胞死亡的机制发生闭锁。尽管促性腺激素释放激素（GnRH）及其激动剂已被证明可抑制垂体切除大鼠的卵巢生长和分化，但关于GnRH诱导卵泡闭锁的研究结果相互矛盾。在本研究中，我们在垂体切除并用雌激素处理的未成熟大鼠中研究了GnRH对卵巢凋亡发生的直接影响。在手术和植入雌激素胶囊2天后，大鼠用GnRH激动剂（GnRHa；[去甘氨酸10，D-苯丙氨酸6，脯氨酸9-N-乙酰胺]GnRH；50微克/注射，每日两次）进行处理。48小时后分离总卵巢DNA，用[32P]双脱氧ATP在3'末端进行标记，并进行大小分级分离。与对照动物相比，用GnRHa处理使180碱基对倍数的DNA片段化增加，这是凋亡的一个标志，表明GnRH直接增加卵巢凋亡性细胞死亡。相反，促卵泡激素（FSH）处理（10微克/注射，每日两次）减少了凋亡性DNA片段化，并且FSH的抗凋亡作用被同时给予GnRHa部分阻断。GnRHa的凋亡诱导作用具有时间和剂量依赖性，处理24小时后可见显著增加，10微克GnRHa/注射时最大增加5.5倍。与使用从整个卵巢分离的DNA的研究相似，从分离的颗粒细胞获得的DNA在GnRHa处理后也显示出DNA片段化的时间和剂量依赖性增加。GnRHa对DNA片段化的作用是由卵巢GnRH受体介导的，因为一种有效的GnRH受体阻滞剂阿扎林B可阻止GnRHa的作用。此外，使用地高辛标记的双脱氧UTP对DNA进行原位末端标记显示，DNA片段化局限于窦前卵泡和窦卵泡的颗粒细胞。在初级卵泡的颗粒细胞或卵泡膜细胞和间质细胞中未检测到GnRHa诱导的凋亡。这些数据表明GnRH直接增加卵巢中的凋亡性细胞死亡，并且GnRH的作用局限于颗粒细胞。这些数据为未来关于卵泡闭锁机制以及GnRH对卵巢核酸内切酶调节的研究提供了基础。
