Cellular Determinants of Differentiation Versus Apoptosis in Avian Granulosa Cells

这项建议描述了促进禽类卵巢颗粒细胞活力的分泌性和非分泌性因子的研究，以及在卵泡选择时促进分化到发育的最后阶段(排卵前等级)的研究。在大多数脊椎动物中，包括禽类在内，绝大多数(90%)正在生长的卵泡在排卵前阶段发育的某个时间点由于卵泡的死亡(卵泡闭锁)而丧失。到目前为止，所有研究的脊椎动物的卵泡闭锁都是通过细胞凋亡来调节的，而且这个过程是在颗粒细胞层开始的。促进颗粒细胞存活并为卵泡的最佳发育提供足够数量的卵泡从而获得最佳生育力的机制的确定，仍然是一个活跃的研究领域。同样，在禽类中精确控制卵泡选择和建立有序的排卵前等级的细胞过程也鲜为人知。在整个雌性鸟类的生殖寿命中，发育中的卵泡的持续磨损最终导致对卵泡刺激素有反应的一小部分卵泡存活。随后，每天从这个队列中选择一个卵泡快速生长并在排卵前进行最终分化，从而建立一个有序的排卵前卵泡等级。值得注意的是，进入排卵前等级的卵泡选择反映在抗凋亡因子和细胞生存信号通路的伴随表达上，这些信号通路使颗粒细胞抵抗凋亡，从而降低了对卵泡闭锁的易感性。因此，在卵泡选择时，卵泡分化的启动与颗粒细胞层内获得的凋亡抵抗之间存在明显的关系。本方案描述了以家鸡卵巢为模型系统的实验，这些实验研究了几种分泌性和非分泌性因子，以及一种独特的凋亡抑制蛋白(IAP)，它们被认为共同调节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、体外分析和分子技术相结合的方法，首席研究员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信号通路，同时抑制细胞的过早分化；2)确定转化生长因子b(TGFb)及其相关家族成员激活素在卵泡选择时促进颗粒细胞分化的作用。相关研究将确定EGF-R信号通路阻断TGFb/激活素诱导分化的机制；以及3)探讨多功能IAP家族成员Survivin在发育成熟的卵泡颗粒细胞中作为细胞增殖调节因子和凋亡抑制因子的作用。这些研究的一个重要成果将是更好地了解促进颗粒细胞对凋亡细胞死亡的抵抗并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理解建立卵巢排卵前卵泡层次的机制，这是包括爬行动物在内的各种产卵(卵生)物种产卵的先决条件。所描述的所有研究都将包括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鉴定，从而有助于进一步鉴定禽类基因组。因此，所有结果都将迅速提交到适当的、可供公众查阅的数据库(如GenBank)，并随后在一线期刊上发表。

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.