ROLE OF MATERNAL PAD16 IN EMBRYONIC DEVELOPMENT

母体 PAD16 在胚胎发育中的作用

• 批准号: 7389541
• 负责人: Scott Alexander Coonrod
• 金额: $ 27.82万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7389541
• 关键词: Anastomosis - action; Antibodies; Arginine; Calcium Signaling; Cell Nucleus; Cells; Chromatin; Citrulline; Competence; Complex; Cytokeratin; Cytoplasm; Cytoskeleton; Defect; Development; Electrons; Embryo; Embryonic Development; Enzymes; Event; Failure; Family; Female; Fertilization; Funding; Genes; Genetic Transcription; Genome; Germ Cells; Growth; Infertility; Keratin; Localized; Mammals; Microscopic; Mus; N-terminal; Nuclear; Oocytes; Phenotype; Play; Protein-arginine deiminase; Proteins; Proteome; RNA-Binding Proteins; Research Personnel; Role; Signal Transduction; Site; Solubility; Staging; Structure; Testing; Time; Triton; Triton X100; base; egg; embryo cell; member; novel; nuclear transfer; research study; transcription factor; zygote

DESCRIPTION (provided by applicant): The mechanisms governing the transition from a terminally differentiated oocyte to a totipotent zygote remain poorly understood. During the previous funding period, we cloned and characterized a highly-abundant oocyte and embryo-restricted gene, peptidylarginine deiminase 6 (PADI6), from the murine egg proteome and found that it represents a novel member of an enzyme family (PADI) that converts protein arginine residues to citrulline. Our ongoing analysis of the PADI6 -/- phenotype indicates that PADI6 -/- females are infertile due to an arrest in embryogenesis at the two-cell stage, thus demonstrating that PADI6 represents a novel maternal effect gene. At the ultrastructural level, PADI6 localizes to an egg and embryo-restricted structure, the cytoskeletal sheets (CSS), that occupies over 15% of the cytoplasm in many mammals. While the function of this cytokeratin-containing structure is unknown, the CSS form during oocyte growth and undergo dramatic reorganizations at critical developmental time points; suggestive of a role in embryonic reprogramming. Strikingly, preliminary ultrastructural analysis reveals a complete dispersal of the cytoskeletal sheets in PADI6 -/- oocytes and eggs while most other structures appear normal. We also found that transcriptional activity in PADI6 -/- two-cell embryos is severely compromised, suggesting that the PADI6 defect arises due to failure to activate embryonic transcription. Based on our preliminary findings, we hypothesize that, in the oocyte, PADI6 interacts with cytokeratin via its N-terminal domain leading to nucleation of the dispersed CSS components. Following fertilization, we hypothesize that PADI6 becomes activated (most likely by calcium signaling) and citrullinates cytokeratin via its C-terminus, leading to CSS reorganization and a stage-dependent release of associated proteins which are required for embryonic genome activation (EGA). The specific aims of this application are to: 1) Test the hypothesis that PADI6 is required for nucleation of CSS components into the mature 60 nm complex in the oocyte. 2) Test the hypothesis that PADI6 plays a role in cytoplasmic-to-nuclear signaling events in the early embryo prior to genome activation. 3) Test the hypothesis that citrullination of cytoskeletal sheet proteins by PADI6 is required for CSS anastomoses and for early development.

描述（由申请人提供）：控制从终末分化卵母细胞向全能受精卵转变的机制仍然知之甚少。在上一个资助期间，我们从小鼠卵蛋白质组中克隆并鉴定了一个高度丰富的卵母细胞和胚胎限制基因肽基精氨酸脱亚胺酶 6 (PADI6)，并发现它代表了将蛋白质精氨酸残基转化为瓜氨酸的酶家族 (PADI) 的新成员。我们对 PADI6 -/- 表型的持续分析表明，PADI6 -/- 雌性由于两细胞阶段胚胎发生停滞而不育，从而证明 PADI6 代表一种新型母体效应基因。在超微结构水平上，PADI6 定位于卵和胚胎限制的结构，即细胞骨架片 (CSS)，它在许多哺乳动物中占据超过 15% 的细胞质。虽然这种含有细胞角蛋白的结构的功能尚不清楚，但 CSS 在卵母细胞生长过程中形成，并在关键的发育时间点经历戏剧性的重组。暗示在胚胎重编程中的作用。引人注目的是，初步超微结构分析显示 PADI6 -/- 卵母细胞和卵子中的细胞骨架片完全分散，而大多数其他结构看起来正常。我们还发现 PADI6 -/- 双细胞胚胎中的转录活性严重受损，表明 PADI6 缺陷是由于未能激活胚胎转录而产生的。根据我们的初步发现，我们假设，在卵母细胞中，PADI6 通过其 N 末端结构域与细胞角蛋白相互作用，导致分散的 CSS 成分成核。受精后，我们假设 PADI6 被激活（很可能是通过钙信号传导）并通过其 C 末端瓜氨酸细胞角蛋白，导致 CSS 重组和胚胎基因组激活 (EGA) 所需的相关蛋白的阶段依赖性释放。该应用的具体目标是： 1) 检验以下假设：CSS 成分成核成卵母细胞中成熟的 60 nm 复合体需要 PADI6。 2) 测试 PADI6 在基因组激活之前的早期胚胎中的细胞质到核信号传导事件中发挥作用的假设。 3) 检验以下假设：CSS 吻合和早期发育需要 PADI6 对细胞骨架片蛋白进行瓜氨酸化。