Control of Oocyte Maturation in C. elegans

描述(申请人提供)：我们的目标是确定线虫中细胞间信号协调减数分裂和受精的机制。在有性生殖的动物中，卵母细胞在双线期或终变期停止，并对激素做出反应，恢复减数分裂(减数分裂成熟)。女性减数分裂I期染色体分离错误是导致人类出生缺陷的主要原因。卵巢荷尔蒙环境中与年龄相关的变化是一个可能的原因。我们的研究建立了线虫作为研究减数分裂成熟的激素控制的遗传范例。线虫精子输出主要精子蛋白(MSP)激素来触发减数分裂成熟。前一个获奖期的一个主要进展是我们发现卵泡样鞘细胞中的G1s-腺苷环化酶信号是所有已知的胚系中依赖MSP的减数分裂成熟反应所必需的。相反，当没有MSP时，VAB-1 MSP/Eph受体在卵母细胞中起着抑制减数分裂成熟的非必要作用。这项应用的具体目的是测试一个体细胞控制减数分裂成熟的模型，在该模型中，鞘细胞控制卵母细胞的反应。目标1定义了鞘细胞如何接收MSP信号。我们将检验MSP与多个G蛋白偶联受体(GPCRs)结合以促进减数分裂成熟的假设。FRET测量将询问MSP是否激活鞘细胞中的G1s-腺苷环化酶途径。一系列的功能测试将检验鞘细胞表达的GPCRs在减数分裂成熟中的作用。在哺乳动物中，保守的MSP结合GPCRs可能介导MSP相关配体的信号传导。目的2阐明鞘细胞如何将MSP信号转导到卵母细胞。我们将通过拮抗两条抑制途径来检验这一假设，即体细胞cAMP依赖的蛋白激酶A促进减数分裂成熟。基因测试将解决在鞘细胞中是否需要PKA来进行减数分裂成熟，以及单个Acy-4抑制基因是否在PKA下游发挥作用。我们将从分子上确定关键的抑制子基因座。这些研究将确定鞘细胞如何将MSP的存在传达给卵母细胞。目的3剖析减数分裂成熟所需的翻译调控机制。保守的锌指蛋白OMA-1和OMA-2(OMA蛋白)是减数分裂成熟所必需的冗余蛋白。蛋白质组学分析表明，OMA蛋白具有转录后基因调节功能。我们将检验这样的假设，即OMA蛋白在没有MSP时抑制减数分裂成熟因子mRNAs的翻译，但当MSP存在时激活翻译。我们将首先使用RNA结合蛋白免疫沉淀-微阵列分析来鉴定OMA核糖核蛋白中的mRNAs。然后，我们将确定OMA蛋白、MSP信号和OMA-核糖核蛋白成分对关键靶标mRNAs表达的影响。这些研究将为减数分裂成熟的翻译调控提供洞察力。 公共卫生相关性： 女性减数分裂I期的染色体分离错误是人类出生缺陷的主要原因，卵巢激素环境的年龄相关变化是一个可能的原因。小的线虫是研究减数分裂激素控制的已建立的模型。这些研究将确定控制雌性减数分裂的信号机制，并对减数分裂错误的起源提供见解。

DESCRIPTION (provided by applicant): Our objective is to define the mechanisms by which intercellular signaling coordinates meiosis and fertilization in the nematode C. elegans. In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects. And age-related changes in the hormonal environment of the ovary are a suggested cause. Our research established C. elegans as a genetic paradigm for studying hormonal control of meiotic maturation. C. elegans sperm export the major sperm protein (MSP) hormone to trigger meiotic maturation. A major advance in the prior award period was our finding that G1s-adenylate cyclase signaling in the follicle-like sheath cells is required for all known MSP-dependent meiotic maturation responses in the germline. By contrast, the VAB-1 MSP/Eph receptor plays a non-essential role in the oocyte to inhibit meiotic maturation when MSP is absent. The Specific Aims of this application test a model for the somatic control of meiotic maturation in which the sheath cells control the oocyte response. AIM 1 defines how the sheath cells receive the MSP signal. We will test the hypothesis that MSP binds multiple G protein-coupled receptors (GPCRs) to promote meiotic maturation. FRET measurements will ask whether MSP activates the G1s-adenylate cyclase pathway in sheath cells. A series of functional tests will examine the role of sheath cell-expressed GPCRs in meiotic maturation. Conserved MSP-binding GPCRs may mediate the signaling of MSP-related ligands in mammals. AIM 2 elucidates how the sheath cells transduce the MSP signal to the oocyte. We will test the hypothesis that somatic cAMP-dependent protein kinase A promotes meiotic maturation by antagonizing two inhibitory pathways. Genetic tests will address whether PKA is required in the sheath cells for meiotic maturation and whether individual acy-4 suppressor loci function downstream of PKA. We will molecularly identify critical suppressor loci. These lines of investigation will define how the sheath cells communicate the presence of MSP to the oocyte. AIM 3 dissects translational control mechanisms required for meiotic maturation. The conserved zinc finger proteins OMA-1 and OMA-2 (OMA proteins) are redundantly required for meiotic maturation. Proteomic analyses suggest that OMA proteins function as post-transcriptional gene regulators. We will test the hypothesis that OMA proteins function to repress translation of meiotic maturation factor mRNAs when MSP is absent, but activate translation when MSP is present. We will first identify mRNAs in OMA- ribonucleoproteins using RNA-binding protein immunoprecipitation-microarray profiling. We will then determine the effects of OMA proteins, MSP signaling, and OMA-ribonucleoprotein components on expression of key target mRNAs. These studies will provide insights into translational regulation of meiotic maturation. PUBLIC HEALTH RELEVANCE: Chromosome segregation errors in female meiosis I are the leading cause of human birth defects and age- related changes in the hormonal environment of the ovary are a suggested cause. The small roundworm C. elegans is an established model for studying the hormonal control of meiosis. These studies will define the signaling mechanisms controlling female meiosis and provide insights into the origin of meiotic errors.