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

总结 成功的胚胎发育依赖于雌配子通过减数分裂正确地进行。 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并进行第一次减数分裂 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 在受精后完成。减数分裂I或II的缺陷损害了卵子的质量和卵子形成一个完整的细胞的能力。 健康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，涉及细胞质网络和卵母细胞皮层。当这些结构性的参与者 确定，现在有必要了解这些肌动蛋白为基础的事件的调节，反过来， 这些过程的失调会出问题。我们对卵母细胞肌动蛋白调节蛋白的分析， 鉴定了p21激活激酶4（PAK 4）在卵母细胞皮层和纺锤体的组织中起关键作用。 由于Rac 1和Cdc 42在卵母细胞中具有关键作用，而PAK 4是由这些Rho家族激活的激酶， 已知PAK 4是GTP酶，并且影响肌动蛋白和微管细胞骨架，PAK 4是一个理想的候选者， 这些GTP酶介导减数分裂I和II中的关键事件的联系。我们发现卵母细胞表达 PAK 4，PAK 4在体外成熟的卵母细胞中与排卵的卵相比是错误定位的。我们也证明了 用PAK 4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， 在中期II，细胞形状扭曲，几种细胞骨架细胞极性标记物异常定位。 虽然这种PAK 4抑制剂可能会影响其他PAK，但PAK 4是我们关注的焦点。 其他PAK在小鼠卵母细胞中检测到的唯一其他PAK是PAK 1，但Pak 1敲除是可育的 PAK 4仅具有细微的表型，而PAK 4在卵母细胞中高度富集，显示出上述不同的表型。 定位在排卵与体外成熟卵中，并且Pak 4缺失小鼠是胚胎致死的。我们现在 我建议研究卵母细胞特异性Pak 4条件性敲除（cKO）模型，因为这将是最好的 分析卵母细胞中PAK 4活性丧失的体内和体外方法。在目标1中，我们将生成一个 卵母细胞特异性Pak 4 cKO系通过Pak 4-floxed小鼠系与Zp 3-Cre系杂交。这个Pak 4- floxed线已成功用于其他cKO。目标1还将评估这些PAK的雌性生育力4 cKO小鼠。在目标2中，我们将分析PAK 4缺陷卵母细胞的表型，测试总体的 假设PAK 4介导细胞骨架和纺锤体组织。我们会检查排卵情况， 通过体内和体外减数分裂的进展，卵母细胞中关键蛋白的定位，以及候选蛋白的表达， 涉及PAK 4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GTP酶到肌动蛋白和纺锤体的完整途径，以及PAK 4在减数分裂中精确点的功能。

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.