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Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic cells.

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

Background information. In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes.Results. We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser 473 is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr 308 is localized on centrosome only. Conclusions.The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation. IntroductionMammalian oocytes are arrested in the dictyate stage of the first meiotic prophase. The dictyate stage is characterized by decondensed chromatin and intense transcription. The preovulatory LH (luteinizing hormone) surge initiates resumption of meiosis, possibly via EGF (epidermal growth factor)-like growth factors, and ends with arrest at MII (metaphase II)

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.