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.

摘要 成功的胚胎发育依赖于雌配子在减数分裂过程中的正确发育。 在包括人类在内的大多数哺乳动物中，卵母细胞进入减数分裂和第一次减数分裂。 成熟，由黄体生成素触发的卵泡内信号启动。第二次减数分裂 在受精时完成。减数分裂I或II的缺陷损害了鸡蛋的质量和卵的能力，形成了 健康的胚胎。特别是减数分裂纺锤体的组织、位置、稳定性和功能的异常 与女性生育能力受损有关。卵母细胞纺锤体的组织和定位由 肌动蛋白，涉及细胞质网络和卵母细胞皮质。当这些结构性的参与者被 现在有必要了解这些基于肌动蛋白的事件的调节，进而了解 这些过程的失调是错误的。我们对卵母细胞中肌动蛋白调节蛋白的分析 研究发现，p21激活的蛋白4(PAK4)在卵母细胞皮质和纺锤体的组织中起关键作用。 由于rac1和cdc42在卵母细胞中起着关键作用，而PAK4是由这些Rho家族激活的一种激酶 GTP酶和已知影响肌动蛋白和微管细胞骨架的PAK4是一个理想的候选者 这些GTP酶在减数分裂I和II中调节关键事件的联系。 PAK4，即PAK4在体外成熟卵母细胞中与排卵卵相比存在定位错误。我们还表明， 用PAK4抑制剂处理卵母细胞在减数分裂I中产生异常的染色质和纺锤体组织， 中期II细胞形态扭曲，多个细胞骨架细胞极性标志物定位异常。 虽然这种PAK4抑制剂有可能影响其他PAK，但PAK4是我们关注的焦点 其他的帕克。在小鼠卵母细胞中唯一检测到的另一种PAK是PAK1，但Ak1基因的敲除是可以生育的 只有微妙的表型，而PAK4在卵母细胞中高度丰富，显示出上述的不同 在排卵和体外成熟的卵子中定位，并且pak4缺失的小鼠是胚胎致死的。我们现在 建议研究卵母细胞特有的Pak4条件性基因敲除(CKO)模型，因为这将是最好的 目的分析体内和体外卵母细胞中PAK4活性丧失的情况。在目标1中，我们将生成一个 卵母细胞特异的Pak4 CKO系通过将Pak4花系小鼠品系与Zp3-Cre品系杂交获得。这是帕克4- 花线已经成功地用于其他cKO。目标1还将评估这些PAK4的女性生育力 CKO小鼠。在目标2中，我们将分析PAK4缺乏的卵母细胞的表型，测试整体 PAK4介导细胞骨架和纺锤体组织的假说。我们将检查排卵， 体内外减数分裂研究进展、关键蛋白在卵母细胞中的定位及候选蛋白 涉及PAK4底物的下游通路。这项研究将揭示这种激酶的功能，即 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GTP酶到肌动蛋白和纺锤体的完整通路，以及PAK4的S在减数分裂的精确点上发挥作用。