Cellular Determinants of Differentiation Versus Apoptosis in Avian Granulosa Cells

该提案描述了对分泌和非分泌因子的研究，这些因子负责促进鸟类卵巢颗粒细胞活力，并促进卵泡选择时分化进入发育的最后阶段（排卵前层次）。在大多数脊椎动物中，包括鸟类物种，绝大多数生长中的卵泡（90%）在排卵前阶段之前的发育过程中的某个时间点通过卵泡死亡（卵泡闭锁）而丢失。目前已证实，迄今为止研究的所有脊椎动物的卵泡闭锁都是通过细胞凋亡介导的，并且这一过程是在颗粒细胞层内启动的。促进颗粒细胞存活并提供足够数量的卵泡以实现最佳卵泡发育从而实现最佳生育力的机制的鉴定仍然是一个活跃的研究领域。 同样，精确控制卵泡选择的细胞过程和鸟类排卵前有序等级的建立也知之甚少。在雌性禽类的整个生殖寿命期间，发育中卵泡的持续消耗最终导致对促卵泡激素有反应的一小群卵泡的存活。 随后，每天从该群组中选择单个卵泡以快速生长并在排卵前进行最终分化，从而建立排卵前卵泡的有序等级。值得注意的是，卵泡选择进入禽排卵前的层次是反映了伴随表达的抗凋亡因子和细胞生存信号通路，使颗粒细胞抗凋亡，因此，在减少的易感性卵泡闭锁。因此，在卵泡选择时，颗粒细胞层内分化的启动和细胞凋亡抗性的获得之间存在明确的关系。 本提案描述了实验，使用国内母鸡（原鸡）的卵巢作为模型系统，研究几个分泌和非分泌的因素，加上一个独特的抑制剂的凋亡蛋白（IAP），这两者一起提出调解颗粒细胞凋亡与细胞存活和分化之间的平衡。利用细胞培养和基因转染、体外试验和分子技术的组合，主要研究者将通过实现以下目标来研究这些过程：1）在细胞内建立负责表皮生长因子受体（EGF-R）介导的促进颗粒细胞增殖和细胞存活的作用的信号传导途径，同时抑制早熟细胞分化; 2）确定转化生长因子B（TGF B）和相关家族成员激活素在卵泡选择时促进颗粒细胞分化中的作用。相关研究将确定EGF-R信号传导阻断TGF β/激活素诱导的分化的机制; 3）研究多功能IAP家族成员生存素作为前层卵泡颗粒细胞中细胞增殖调节因子和细胞凋亡抑制因子的作用。这些研究的一个重要结果将是更好地理解促进颗粒细胞抵抗凋亡细胞死亡和促进颗粒细胞分化的致病因素和相关细胞事件。更广泛地说，所产生的数据将有助于我们了解负责建立卵巢排卵前卵泡层次结构的机制，这是各种产卵（卵生）物种（包括爬行动物）产卵的先决条件。 所描述的所有研究都将包括对本科生和研究生的培训。预计在研究过程中，新的禽类基因将被鉴定和功能表征，从而有助于禽类基因组的进一步表征。因此，所有结果将及时提交给适当的、可公开访问的数据库（例如，GenBank），并随后在一级期刊上发表。

This proposal describes studies of secreted and nonsecreted factors responsible for promoting avian ovarian granulosa cell viability, and facilitating differentiation at the time of ovarian follicle selection into the final stages of development (the preovulatory hierarchy). In most vertebrates, including avian species, the vast majority of growing ovarian follicles ( 90%) are lost via the death of follicles (follicle atresia) at some point during development prior to the preovulatory stage. It is now well established that ovarian follicle atresia in all vertebrate species studied to date is mediated via apoptosis, and that this process is initiated within the granulosa cell layer. The identification of mechanisms that promote the survival of granulosa cells and provide for sufficient numbers of ovarian follicles for optimal follicle development and thus, optimal fertility, remains an active area of investigation. Similarly, cellular processes that precisely control ovarian follicle selection and the establishment of an orderly preovulatory hierarchy in avian species are poorly understood. Throughout the reproductive life span of the avian female, ongoing attrition of developing follicles eventually results in the survival of a small cohort of follicles that are responsive to follicle stimulating hormone. Subsequently, a single follicle from this cohort is selected per day to rapidly grow and undergo final differentiation before ovulation, thus establishing an orderly hierarchy of preovulatory follicles. Significantly, follicle selection into the avian preovulatory hierarchy is reflected by the concomitant expression of anti-apoptotic factors and cell survival signaling pathways that render granulosa cells resistant to apoptosis, and accordingly, results in diminished susceptibility to follicle atresia. Thus, there is a clear relationship between the initiation of differentiation and acquisition of apoptosis resistance within the granulosa cell layer at the time of follicle selection. The present proposal describes experiments, using the ovary of the domestic hen (Gallus gallus) as a model system, which study several secreted and nonsecreted factors, plus a unique Inhibitor of Apoptosis Protein (IAP), which together are proposed to mediate a balance between granulosa cell apoptosis versus cell survival and differentiation. Utilizing a combination of cell culture and gene transfection, in vitro assays and molecular techniques, the principal investigator will approach the study of these processes by achieving the following objectives: 1) establish signaling pathways within the cell that are responsible for epidermal growth factor receptor- (EGF-R-) mediated effects on promoting granulosa cell proliferation and cell survival, while simultaneously inhibiting premature cell differentiation; 2) define the role of transforming growth factor b (TGFb) and the related family member, activin, in promoting granulosa cell differentiation at the time of follicle selection. Related studies will define mechanisms by which EGF-R signaling blocks TGFb/activin-induced differentiation; and 3) investigate the role of the multifunctional IAP family member, survivin, as a regulator of cell proliferation and inhibitor of apoptosis in prehierarchal follicle granulosa cells. An important outcome of these studies will be a better understanding of causative factors and associated cellular events that promote granulosa cell resistance to apoptotic cell death and facilitate granulosa cell differentiation. More broadly, the data generated will contribute to our understanding of mechanisms responsible for the establishment of an ovarian preovulatory follicle hierarchy that is prerequisite for egg laying in a variety of egg-laying (oviparous) species, including reptiles. All studies described will incorporate the training of both undergraduate and graduate students. It is expected that during the course of investigations novel avian genes will be identified and functionally characterized, thus contributing to the further characterization of the avian genome. Accordingly, all results will be promptly submitted to appropriate, publicly accessible databases (e.g., GenBank) and subsequently published in first-tier journals.