APOPTOSIS AND REPRODUCTIVE AGING OF THE FEMALE

女性的细胞凋亡和生殖衰老

基本信息

批准号：
2001548
负责人：
Jonathan Lee Tilly
金额：
$ 15.8万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-09-15 至 1998-12-31
项目状态：
已结题

项目摘要

Although depletion of the ovarian follicle pool plays a critical role in reproductive aging in the human female, the events underlying follicular atresia remain to be elucidated. It is well-established that apoptosis, a form of physiological cell death, is important for remodeling of tissues and removal of transient cell populations. Moreover, the penultimate controlling events which initiate or suppress physiological cell death may involve a conserved cohort of "cell death" proteins, and the fate of cells may be decided by the ratio of death repressors (Bcl-2, Bcl-x/long) to death inducers (Bax, Bcl-x/short). The mechanisms of action of these proteins have not been fully clarified. However, there is evidence that Bcl-2 functions as an antioxidant to protect cells from damage by reactive oxygen species such as superoxide anion, peroxide, and hydroxyl radical. It is possible that these death repressor proteins (Bcl-2, Bcl-x/long) are components of a family of oxidative stress response proteins which also includes superoxide dismutase (SOD; an enzyme which catalyzes conversion of superoxide anion to hydrogen peroxide) and glutathione peroxidase (GSHPx; an enzyme which converts hydrogen peroxide to water). As recent studies conducted by the PI have revealed that granulosa cell death during atresia in all species studied thus far, including humans, is mediated via apoptosis, it is hypothesized that follicular atresia is initiated by a shift in the ratio of death repressor to death inducer gene expression. The altered expression of these cell death genes would then lead to insufficient protection of granulosa cells from the damaging effects of reactive oxygen species. To test this hypothesis, the Specific Aims of this proposal are to: 1) characterize changes in the levels of mRNA encoding bcl-2, bax, bcl-x (long and short), SOD and GSHPx during maturation of rat ovarian follicles from the primordial to antral stage of development; 2) evaluate if gonadotropin-mediated inhibition of apoptosis in granulosa cells of rat antral follicles in vivo and in vitro is associated with enhanced bcl-2, bcl-x/long, SOD and GSHPx, and suppressed bax and bcl-x/short, gene expression; 3) determine if selective destruction of the bcl-2, bcl- x/long, SOD and GSHPx genes by the apoptotic endonuclease contributes to a loss of death repressor gene expression during granulosa cell apoptosis and atresia; 4) investigate if inhibitors of oxidative free radical damage prevent apoptosis in granulosa cells of rat antral follicles incubated in vitro without trophic hormone support; and, 5) determine if inducers of oxidative free radical damage interfere with the ability of gonadotropins to inhibit apoptosis in granulosa cells of rat antral follicles incubated in vitro.

尽管卵巢卵泡池的耗竭在人类女性的生殖衰老，卵泡潜在的事件闭锁仍有待阐明。众所周知，细胞凋亡，生理细胞死亡的一种形式，对于重塑组织和去除瞬时细胞群。此外，启动或抑制生理的倒数第二个控制事件细胞死亡可能涉及一组保守的“细胞死亡”蛋白，并且细胞的命运可能由死亡抑制因子（Bcl-2、 Bcl-x/长）至死亡诱导剂（Bax、Bcl-x/短）。其机制这些蛋白质的作用尚未完全阐明。然而，有证据表明 Bcl-2 具有抗氧化剂的功能，可以保护细胞免受活性氧物质如超氧阴离子、过氧化物等造成的损伤羟基自由基。这些死亡抑制蛋白可能（Bcl-2、Bcl-x/long）是氧化应激家族的组成部分反应蛋白，其中还包括超氧化物歧化酶（SOD；一种催化超氧阴离子转化为氢的酶过氧化物）和谷胱甘肽过氧化物酶（GSHPx；一种将过氧化氢到水）。 PI 最近进行的研究表明揭示了所有研究物种闭锁期间颗粒细胞死亡到目前为止，包括人类在内，是通过细胞凋亡介导的，假设卵泡闭锁是由死亡率的变化引起的死亡诱导基因表达的阻遏物。改变后的表达这些细胞死亡基因将导致细胞保护不足颗粒细胞免受活性氧的破坏作用。为了检验这一假设，该提案的具体目标是：1) 表征编码 bcl-2、bax、bcl-x 的 mRNA 水平的变化（长和短），大鼠卵巢成熟过程中的SOD和GSHPx 卵泡从原始发育阶段到窦状阶段； 2）评估如果促性腺激素介导的颗粒细胞凋亡抑制大鼠窦卵泡在体内和体外的增强与 bcl-2、bcl-x/long、SOD 和 GSHPx，以及抑制的 bax 和 bcl-x/short，基因表达； 3) 确定是否选择性破坏bcl-2、bcl- x/long、SOD 和 GSHPx 基因通过凋亡核酸内切酶促进颗粒细胞凋亡过程中死亡抑制基因表达缺失和闭锁； 4) 研究氧化自由基抑制剂是否损伤防止大鼠窦卵泡颗粒细胞凋亡在没有营养激素支持的情况下进行体外培养；以及，5) 确定是否氧化自由基损伤的诱导剂会干扰促性腺激素抑制大鼠胃窦颗粒细胞凋亡体外培养的卵泡。