p21-activated kinase as regulator of actin and microtubules in mammalian oocytes

p21 激活激酶作为哺乳动物卵母细胞肌动蛋白和微管的调节剂

• 批准号: 9387058
• 负责人: JANICE P EVANS
• 金额: $ 8.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387058
• 关键词: Actins; Affect; Assisted Reproductive Technology; Cell Polarity; Cell Shape; Cell division; Chromatin; Competence; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Embryo; Embryonic Development; Event; F-Actin; Family; Female; Female infertility; Fertility; Fertilization; Foundations; Future; G Actin; Germ Cells; Guanosine Triphosphate Phosphohydrolases; Human; Impairment; In Vitro; Jordan; Knock-out; Knockout Mice; LIM Domain; LIMK1 gene; Letters; Link; Litter Size; LoxP-flanked allele; Luteinizing Hormone; Mammals; Mediating; Meiosis; Meiotic Prophase I; Metaphase; Microfilaments; Microtubules; Modeling; Mus; Oocytes; Outcome; Ovulation; Pathway interactions; Phenotype; Phosphotransferases; Positioning Attribute; Pregnancy; Process; Proteins; Regulation; Reproduction; Research; Signal Transduction; Testing; Time; Work; base; cell type; cofilin; depolymerization; egg; genetic regulatory protein; in vivo; inhibitor/antagonist; knockout gene; oocyte maturation; p21 activated kinase; public health relevance; pup; reproductive; rho; treatment effect

SUMMARY Successful embryonic development is dependent on the female gamete progressing correctly through meiosis. In most mammals including human, entry into meiosis and the first meiotic division occur with oocyte maturation, initiated by intrafollicular signals triggered by luteinizing hormone. The second meiotic division completes upon fertilization. Defects in meiosis I or II compromise egg quality and egg competence to form a healthy embryo. In particular, abnormal organization, positioning, stability, and function of the meiotic spindles are associated with impaired female fertility. Oocyte spindle organization and positioning are orchestrated by actin, involving a cytoplasmic meshwork and the oocyte cortex. As these structural players are being identified, there is now the need to understand the regulation of these actin-based events and, in turn, what goes wrong with dysregulation of these processes. Our analysis of actin-regulatory proteins in oocytes has identified p21-activated kinase 4 (PAK4) as a key player in organization of the oocyte cortex and spindle. Since Rac1 and Cdc42 have key roles in oocytes, and PAK4 is a kinase activated by these Rho-family GTPases and known to affect both the actin and microtubule cytoskeletons, PAK4 is an ideal candidate to be the link from these GTPases to mediate crucial events in meiosis I and II. We show that oocytes express PAK4, that PAK4 is mislocalized in in vitro matured oocytes as compared to ovulated eggs. We also show that treatment of oocytes with a PAK4 inhibitor produces abnormal chromatin and spindle organization in meiosis I, and distorted cell shape and abnormal localization of several cytoskeletal cell polarity markers at metaphase II. While there is the formal possibility that this PAK4 inhibitor could affect other PAKs, PAK4 is our focus over other PAKs. The only other PAK to be detected in mouse oocytes is PAK1, but the Pak1 knockout is fertile with only subtle phenotypes, whereas PAK4 is highly enriched in oocytes, shows the aforementioned different localization in ovulated versus in vitro matured eggs, and the Pak4 null mouse is embryonic lethal. We now propose to study an oocyte-specific Pak4 conditional knockout (cKO) model, as this will be the best approach to analyze the loss of PAK4 activity in oocytes, in vivo and in vitro. In Aim 1, we will generate an oocyte-specific Pak4 cKO line by crossing the Pak4-floxed mouse line with the Zp3-Cre line. This Pak4- floxed line has been used successfully for other cKOs. Aim 1 also will assess female fertility of these PAK4 cKO mice. In Aim 2, we will analyze the phenotypes of PAK4-deficient oocytes, testing the overall hypothesis that PAK4 mediates cytoskeletal and spindle organization. We will examine ovulation, progression through meiosis in vivo and in vitro, the localizations of key proteins in oocytes, and candidate downstream pathways involving PAK4 substrates. This research will reveal the functions of this kinase that is positioned to be a key player in oocytes, and provide the foundation for future studies, including analysis of the full pathway from GTPases to actin and spindle, and of PAK4's functions at precise points in meiosis.

概括 成功的胚胎发育取决于雌配子通过减数分裂的正确进展。 在包括人类在内的大多数哺乳动物中，进入减数分裂和第一次减数分裂是由卵母细胞发生的 成熟，由促黄体激素触发的卵泡内信号启动。第二次减数分裂 受精后完成。减数分裂 I 或 II 的缺陷会损害卵子质量和卵子形成能力 健康的胚胎。特别是减数分裂纺锤体的异常组织、定位、稳定性和功能 与女性生育能力受损有关。卵母细胞纺锤体的组织和定位是由 肌动蛋白，涉及细胞质网和卵母细胞皮质。由于这些结构性参与者正在 确定后，现在需要了解这些基于肌动蛋白的事件的调节，以及反过来，什么是 这些过程的失调就会出现问题。我们对卵母细胞中肌动蛋白调节蛋白的分析 确定 p21 激活激酶 4 (PAK4) 是卵母细胞皮质和纺锤体组织的关键参与者。 由于 Rac1 和 Cdc42 在卵母细胞中具有关键作用，而 PAK4 是由这些 Rho 家族激活的激酶 GTP 酶已知会影响肌动蛋白和微管细胞骨架，PAK4 是理想的候选者 这些 GTP 酶与介导减数分裂 I 和 II 中关键事件的联系。我们证明卵母细胞表达 PAK4，与排卵的卵相比，PAK4 在体外成熟的卵母细胞中错误定位。我们还表明 用 PAK4 抑制剂处理卵母细胞会在减数分裂 I 中产生异常染色质和纺锤体组织， 以及中期 II 细胞形状扭曲和多个细胞骨架细胞极性标记的异常定位。 虽然这种 PAK4 抑制剂可能会影响其他 PAK，但 PAK4 是我们关注的重点 其他 PAK。在小鼠卵母细胞中检测到的唯一其他 PAK 是 PAK1，但 Pak1 敲除是可育的 仅具有微妙的表型，而 PAK4 在卵母细胞中高度富集，显示了上述不同的 排卵与体外成熟卵中的定位，并且 Pak4 缺失小鼠是胚胎致死的。我们现在 建议研究卵母细胞特异性 Pak4 条件敲除 (cKO) 模型，因为这将是最好的 分析卵母细胞体内和体外 PAK4 活性丧失的方法。在目标 1 中，我们将生成一个 通过将 Pak4-floxed 小鼠系与 Zp3-Cre 系杂交，获得卵母细胞特异性 Pak4 cKO 系。这个Pak4- floxed 线已成功用于其他 cKO。目标 1 还将评估这些 PAK4 的女性生育能力 cKO小鼠。在目标 2 中，我们将分析 PAK4 缺陷卵母细胞的表型，测试整体 PAK4 介导细胞骨架和纺锤体组织的假设。我们会检查排卵， 体内和体外减数分裂的进展、卵母细胞中关键蛋白的定位以及候选蛋白 涉及 PAK4 底物的下游途径。这项研究将揭示这种激酶的功能 定位为卵母细胞的关键参与者，并为未来的研究提供基础，包括分析 从 GTPases 到肌动蛋白和纺锤体的完整途径，以及 PAK4 在减数分裂精确点的功能。