Robles R, Tao X J, Trbovich A M, Maravel D V, Nahum R, Perez G I, Tilly K I, Tilly J L
Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston 02114-2696, USA.
Endocrinology. 1999 Jun;140(6):2641-4. doi: 10.1210/endo.140.6.6931.
The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bcl-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.
近期在脊椎动物中鉴定出凋亡蛋白酶激活因子1（Apaf-1）是秀丽隐杆线虫基因ced-4的假定同源物，这表明秀丽隐杆线虫程序性细胞死亡机制的第三个主要分支在进化过程中也得到了保留。尽管现在已知细胞凋亡对脊椎动物的卵巢卵泡闭锁很重要，但对于Apaf-1在卵巢功能中的作用却一无所知。在此我们通过免疫组织化学分析表明，Apaf-1在早期有腔卵泡的颗粒细胞中大量存在，而体内促性腺激素预处理完全抑制了Apaf-1的表达和颗粒细胞凋亡。对在无激素支持下体外培养以诱导凋亡前后的颗粒细胞制备的分级蛋白提取物进行蛋白质印迹分析表明，线粒体细胞色素c释放这一Apaf-1激活所需的生化步骤发生在体外培养的颗粒细胞中。此外，对procaspase-3加工的蛋白质印迹分析，这是由活化的Apaf-1引发的主要下游事件，表明健康的颗粒细胞几乎只拥有该酶的无活性（前体）形式，而缺乏激素支持的颗粒细胞会迅速将procaspase-3加工成活性酶。最后，我们通过单细胞荧光半胱天冬酶活性测定表明，血清饥饿的颗粒细胞在核固缩之前和之后都会激活半胱天冬酶3样酶。这些数据，结合先前关于秀丽隐杆线虫另外两个细胞死亡调节基因ced-9（Bcl-2家族成员）和ced-3（半胱天冬酶）的同源物在闭锁中的作用的观察结果，充分支持了这样的假设，即颗粒细胞凋亡是由进化中保留的细胞死亡程序的所有三个主要分支精确协调的。
