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

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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.