PILOT PROJECT: Regulation of Translational Control in the Oocyte-Embryo Transiti

B.意义 卵母细胞成熟和从分化生殖细胞向全能单细胞受精的转变 受精卵标志着后生动物发展的开始。了解最早的阶段 发育，特别是卵母细胞向胚胎的转变，对生殖至关重要。 医药。人类生殖细胞和植入前胚胎的缺陷会导致临床不孕、流产和 还有潜在的先天缺陷。我们的发现，以及开发的方法，将提供强大的， 功能丰富、带注释的数据集，不仅将成为生殖系统的宝贵资源 医学/生物学社区，但也将有助于将生殖生物学带到广泛的前沿 科学观众。 A.背景和具体目标 大多数，如果不是全部，哺乳动物初始发育所需的mRNAs和蛋白质 胚胎来自卵母细胞，即女性生殖细胞。受精的单细胞受精卵被认为是 转录上的沉默。母性因素支持和指导最早的发展阶段，包括 第一次有丝分裂细胞周期(S)和核重新编程，至少直到胚胎基因组被激活。这个 胚胎基因组激活(EGA)的精确时间在哺乳动物物种中是不同的。在人类中，EGA EGA在4到8细胞阶段开始，而在小鼠中，EGA在2细胞阶段结束时发生[1]。 由于在EGA之前没有或极少的胚胎转录，胚胎依赖于各种转录后转录 初步发展的机制。特别是，以前的工作已经表明，对 在卵母细胞到胚胎的转变过程中，mRNA的翻译可能是至关重要的。在发育中的卵母细胞中，一些mRNAs 去烯化并储存在卵质中以备以后翻译。未成熟卵母细胞去烯基化的mRNAs 被含有非活性CPEB的蛋白质复合体结合并保持沉默，这是一种高度保守的RNA结合 促进mRNAs多聚腺嘌呤尾部伸长的蛋白质，5‘帽结合因子elF4E，以及 Maskin，这是一种与elF4E相互作用并阻止elF4E招募其他5‘帽元件的蛋白质 对于翻译的启动来说是必要的。在卵母细胞成熟时，一系列分子事件导致添加 将数百个腺嘌呤残基连接到3‘末端，并将Maskin从elF4E解离，以允许翻译。 在这一过程中，至少有两个c/S元件是必需的，即核多聚腺苷酸化信号和核多聚腺苷酸化信号。 胞质多聚腺苷酸化元件(CPE)。转录本在两细胞胚胎中异常大的转录本 在其3‘UTRs中包含CPE序列[2]。 理解卵母细胞到胚胎转变过程中转录后和翻译的控制是一种 这是我们实验室研究的重要和新方向。我们最近发现母版转录 因子Oct4可能起到将早期胚胎发育程序从依赖于 转录后控制主要由转录网络调控[3]。这一潜力 Oct4的新作用有别于其已确立的调节胚胎多能性的关键功能 并将体细胞重新编程为更具多能性的状态。当我们在10月4日之前 注射吗啉寡核苷酸(MOS)，可同时沉默母体和胚胎 转录迅速，进入单细胞受精卵，我们发现大部分胚胎停滞在多细胞阶段。 值得注意的是，我们发现两细胞胚胎中Oct4调节的基因富含翻译(例如， 真核细胞翻译起始因子(Elf)和elF4E2或4EHP，通过与5‘端结合来抑制翻译 帽而不是ELF)和RNA处理功能。果蝇elF4E2或d4EHP的同源物可能 可能与Rbp4相互作用，Rbp4是翻译抑制细胞周期蛋白B所必需的(见 富勒实验室)。 这些观察表明转录后控制是一种高度保守和关键的 卵母细胞向胚胎转化的机制。然而，在哺乳动物模型中，对它的了解仍然很少。 因为以前关于小鼠卵母细胞向胚胎转化的研究大多是基于候选基因 他说，这是一种可行的方法，进展相当缓慢。随着基因组学技术的出现，我们现在可以开始 以高通量的方式询问重要的生物学问题。 假设：我们假设至少某些基因转录后和翻译后的控制是 在卵母细胞到胚胎的转变过程中至关重要，并由Oct4控制，Oct4是 重新编程和多能性。在试点项目中，我们建议采用下一代测序(NGS) 了解卵母细胞向胚胎转化过程中转录后调控和翻译调控的程度 并确定到10月4日，这种控制在多大程度上是通过中介实现的。

B. SIGNIFICANCE Oocyte maturation and the transition from differentiated germ cells to a totipotent one-cell fertilized zygote mark the beginning of the development of a metazoan. Understanding the earliest stages of development, particularly the oocyte-to-embryo transition, is of fundamental importance to reproductive medicine. Defects in human germ cells and preimplantation embryos lead to clinical infertility, miscarriage, and potentially birth defects as well. Our findings, along with methodologies that are developed, will provide robust, functionally-rich, and annotated datasets that will not only be valuable resources for the reproductive medicine/biology communities, but will also serve to bring reproductive biology to the forefront of the broad scientific audience. A. BACKGROUND AND SPECIFIC AIMS Most, if not all, of the mRNAs and proteins required for the initial development of the mammalian embryo come from the oocyte, the female germ cell. The fertilized, one-cell zygote is thought to be transcriptionally silent. Maternal factors support and direct the earliest stages of development, including the first mitotic cell cycle(s) and nuclear reprogramming, at least until the embryonic genome is activated. The precise timing of embryonic genome activation (EGA) varies among mammalian species. In humans, EGA initiates at the 4- to 8-cell stages, while in the mouse, EGA occurs by the end of the 2-cell stage[1]. Since there is no or minimal embryonic transcription prior to EGA, the embryo relies on various posttranscriptional mechanisms for initial development. In particular, previous work has indicated that control of mRNA translation may be crucial for the oocyte-to-embryo transition. In the growing oocyte, some mRNAs are deadenylated and stored in the ooplasm for later translation. The deadenylated mRNAs of immature oocytes are bound and kept silent by protein complexes containing inactive CPEB, which is a highly conserved RNA-binding protein that promotes elongation of the polyadenine tail of mRNAs, the 5' cap-binding factor elF4E, and maskin, which is a protein that interacts with and prevents elF4E from recruiting other 5' cap elements necessary for translation initiation. Upon oocyte maturation, a series of molecular events results in the addition of several hundred adenine residues to the 3' tail and dissociation of maskin from elF4E to allow for translation. At least two c/s-elements are necessary for this process, namely the nuclear polyadenylation signal and the cytoplasmic polyadenylation element (CPE). An unusually large fraction of transcripts in the two-cell embryo contains CPE sequences in their 3'UTRs[2]. Understanding post-transcriptional and translational control during the oocyte-to-embryo transition is an important and new direction in our laboratory's research. We recently discovered that the master transcription factor Oct4 may function to switch the early embryonic developmental program from one that is dependent on post-transcriptional control to one that is predominantly regulated by a transcriptional network[3]. This potential new role of Oct4 is distinct from its well established and critical function to regulate pluripotency in embryonic stem cells and to reprogram somatic cells into a more pluripotent state. When we knocked down Oct4 by injecting morpholino oligonucleotides (MOs), which could simultaneously silence both maternal and embryonic transcripts rapidly, into the one-cell zygote, we found that most of the embryos arrested by the multicell stage. Significantly, we found that Oct4-regulated genes in the two-cell embryo were enriched for translation (e.g. eukaryotic translation initiation factors (elFs) and elF4E2 or 4EHP, which inhibits translation by binding to the 5' cap but not to the elFs) and RNA processing functions. The Drosophila homolog of elF4E2, or d4EHP, may potentially interact with Rbp4, which is required for translational repression of cyclin B (see Project 3 from the Fuller laboratory). These observations suggest that post-transcriptional control is a highly conserved and critical mechanism for the oocyte-to-embryo transition. However, it remains poorly understood in mammalian models because most of the previous studies on mouse oocyte-to-embryo transition were based on a candidate gene approach and progressed considerably slowly. With the advent of genomics technologies, we can now begin to interrogate important biological questions in a high throughput manner. Hypothesis: We hypothesize that post-transcriptional and translational control of at least some genes is critical during the oocyte-to-embryo transition, and is controlled by Oct4, the master regulator of reprogramming and pluripotency. In the pilot project, we propose to adopt next generation sequencing (NGS) to understand the extent of post-transcriptional and translational control during the oocyte-to-embryo transition and to determine how much of this control is mediated by Oct4.