GSK3 beta mediates acentromeric spindle stabilization by activated PKC zeta

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKCζ (p-PKCζ) and Glycogen Synthase Kinase 3β (GSK3β) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKCζ (p-PKCζ) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3β and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKCζ at the meiotic spindle. When GSK3β is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3β is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKCζ maintains spindle organization appears to be through GSK3β and suggests that p-PKCζ phosphorylates GSK3β on the ser9 position inactivating GSK3β and consequently maintaining spindle stability during meiotic metaphase arrest.     

The Caenorhabditis elegans Aurora B kinase AIR-2 phosphorylates and is required for the localization of a BimC kinesin to meiotic and mitotic spindles

BimC kinesins are required for mitotic spindle assembly in a variety of organisms. These proteins are localized to centrosomes, spindle microtubules, and the spindle midzone. We have previously shown that the Caenorhabditis elegans Aurora B kinase AIR-2 is required for the localization of the ZEN-4 kinesin protein to midzone microtubules. To determine whether the association of BimC kinesins with spindle microtubules is also dependent on AIR-2, we examined the expression pattern of BMK-1, a C. elegans BimC kinesin, in wild-type and AIR-2-deficient embryos. BMK-1 is highly expressed in the hermaphrodite gonad and is localized to meiotic spindle microtubules in the newly fertilized embryo. In mitotic embryos, BMK-1 is associated with spindle microtubules from prophase through anaphase and is concentrated at the spindle midzone during anaphase and telophase. In the absence of AIR-2, BMK-1 localization to meiotic and mitotic spindles is greatly reduced. This is not a consequence of loss of ZEN-4 localization because BMK-1 is appropriately localized in ZEN-4-deficient embryos. Furthermore, AIR-2 and BMK-1 directly interact with one another and the C-terminal tail domain of BMK-1 is specifically phosphorylated by AIR-2 in vitro. Together with our previous data, these results suggest that at least one function of the Aurora B kinases is to recruit spindle-associated motor proteins to their sites of action.     

Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.

BACKGROUND: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified. RESULTS: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCalpha was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCdelta in vivo. The most highly purified hydrophobic site kinase preparations ( approximately 10,000-fold) reacted with antibodies to PKCzeta/iota. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCzeta from the membrane fraction of transfected cells. An activated mutant of PKCzeta, but not wild-type PKCzeta, induced phosphorylation of the PKCdelta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCzeta mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCzeta could phosphorylate the PKCdelta hydrophobic site in vitro, whereas wild-type PKCzeta could not. CONCLUSIONS: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.     

Anillin localization suggests distinct mechanisms of division plane specification in mouse oogenic meiosis I and II

Anillin is a conserved cytokinetic ring protein implicated in actomyosin cytoskeletal organization and cytoskeletal-membrane linkage. Here we explored anillin localization in the highly asymmetric divisions of the mouse oocyte that lead to the extrusion of two polar bodies. The purposes of polar body extrusion are to reduce the chromosome complement within the egg to haploid, and to retain the majority of the egg cytoplasm for embryonic development. Anillin's proposed roles in cytokinetic ring organization suggest that it plays important roles in achieving this asymmetric division. We report that during meiotic maturation, anillin mRNA is expressed and protein levels steadily rise. In meiosis I, anillin localizes to a cortical cap overlying metaphase I spindles, and a broad ring over anaphase spindles that are perpendicular to the cortex. Anillin is excluded from the cortex of the prospective first polar body, and highly enriched in the cytokinetic ring that severs the polar body from the oocyte. In meiosis II, anillin is enriched in a cortical stripe precisely coincident with and overlying the meiotic spindle midzone. These results suggest a model in which this cortical structure contributes to spindle re-alignment in meiosis II. Thus, localization of anillin as a conserved cytokinetic ring marker illustrates that the geometry of the cytokinetic ring is distinct between the two oogenic meiotic cytokineses in mammals.     

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.     

Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.     

Distinct protein kinase C isoforms mediate regulation of vascular endothelial growth factor expression by A2A adenosine receptor activation and phorbol esters in pheochromocytoma PC12 cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis during development and in disease. In pheochromocytoma (PC12) cells, VEGF expression is regulated by A(2A) adenosine receptor (A(2A)AR) activation. The present work examines the underlying signaling pathway. The adenylyl cyclase-protein kinase A cascade has no role in the down-regulation of VEGF mRNA induced by the A(2A)AR agonist, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKCalpha, PKCepsilon, and PKCzeta, and CGS21680 activated PKCepsilon and PKCzeta as assessed by cellular translocation. By 6 h, PMA but not CGS21680 decreased PKCalpha and PKCepsilon expression. Neither compound affected PKCzeta levels. Following prolonged PMA treatment to down-regulate susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132, abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA. Phorbol 12,13-diacetate reduced VEGF mRNA levels while down-regulating PKCepsilon but not PKCalpha expression. In cells expressing a dominant negative PKCzeta construct, CGS21680 was unable to reduce VEGF mRNA. Together, the findings suggest that phorbol ester-induced down-regulation of VEGF mRNA occurs as a result of a reduction of PKCepsilon activity, whereas that mediated by the A(2A)AR occurs following deactivation of PKCzeta.     

Involvement of the PKC family in regulation of early development

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11‐member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition. Mol. Reprod. Dev. 77: 95–104, 2010. © 2009 Wiley‐Liss, Inc.     

Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization

Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.     

Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked

In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.     

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.     

The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy in cyclin destruction and anaphase progression in meiosis I and II in Drosophila

Meiosis is a highly specialized cell division that requires significant reorganization of the canonical cell-cycle machinery and the use of meiosis-specific cell-cycle regulators. The anaphase-promoting complex (APC) and a conserved APC adaptor, Cdc20 (also known as Fzy), are required for anaphase progression in mitotic cells. The APC has also been implicated in meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. Here, we show that Cort functions together with the canonical mitotic APC adaptor Fzy to target the three mitotic cyclins (A, B and B3) for destruction in the egg and drive anaphase progression in both meiotic divisions. In addition to controlling cyclin destruction globally in the egg, Cort and Fzy appear to both be required for the local destruction of cyclin B on spindles. We find that cyclin B associates with spindle microtubules throughout meiosis I and meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and Cort are required for this loss of cyclin B from the meiotic spindle. Our results lead to a model in which the germline-specific APC(Cort) cooperates with the more general APC(Fzy), both locally on the meiotic spindle and globally in the egg cytoplasm, to target cyclins for destruction and drive progression through the two meiotic divisions.     

Kinesin-6 family motor KIF20A regulates central spindle assembly and acrosome biogenesis in mouse spermatogenesis

Kinesin-6 KIF20A is essential for microtubule organization and central spindle assembly during cytokinesis. However, the functions of KIF20A in meiotic division and spermatogenesis remain elusive. Here, we report that kinesin-6 KIF20A locates at the microtubules in mouse spermatogenic cells and co-localizes with the spindle midzone and midbody. We demonstrate that central spindle organization and chromosomal stability are regulated by KIF20A in male meiotic division. KIF20A inhibition leads to the defects in central spindle assembly and cytokinetic abscission, and finally results in the increase of aneuploid cells and the alteration of cell populations in the spermatogenic cells. Furthermore, we have revealed that kinesin-6 KIF20A is associated with the formation and maturation of the acrosomes during spermatogenesis. Our findings have identified the specific roles of KIF20A in central spindle organization in meiotic division.     

NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.     

Cytokinesis and Midzone Microtubule Organization in Caenorhabditis elegans Require the Kinesin-like Protein ZEN-4

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele of zen-4, an MKLP1 homologue in the nematode Caenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.     

Probing the dynamics and functions of aurora B kinase in living cells during mitosis and cytokinesis

Aurora B is a protein kinase and a chromosomal passenger protein that undergoes dynamic redistribution during mitosis. We have probed the mechanism that regulates its localization with cells expressing green fluorescent protein (GFP)-tagged wild-type or mutant aurora B. Aurora B was found at centromeres at prophase and persisted until approximately 0.5 min after anaphase onset, when it redistributed to the spindle midzone and became concentrated at the equator along midzone microtubules. Depolymerization of microtubules inhibited the dissociation of aurora B from centromeres at early anaphase and caused the dispersion of aurora B from the spindle midzone at late anaphase; however, centromeric association during prometaphase was unaffected. Inhibition of CDK1 deactivation similarly caused aurora B to remain associated with centromeres during anaphase. In contrast, inhibition of the kinase activity of aurora B appeared to have no effect on its interactions with centromeres or initial relocation onto midzone microtubules. Instead, kinase-inactive aurora B caused abnormal mitosis and deactivation of the spindle checkpoint. In addition, midzone microtubule bundles became destabilized and aurora B dispersed from the equator. Our results suggest that microtubules, CDK1, and the kinase activity each play a distinct role in the dynamics and functions of aurora B in dividing cells.     

Effect of age, GV transfer and modified nucleocytoplasmic ratio on PKCα in mouse oocytes and early embryos

Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6-8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.     

Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function

The mature mammalian oocyte is highly polarized because asymmetrical spindle migration to the oocyte cortex ensures extrusion of small polar bodies in the two meiotic divisions, essential for generation of the large egg. Actin filaments, myosin motors, and formin-2, but not microtubules, are required for spindle migration. Here, we show that Cdc42, a key regulator of cytoskeleton and cell polarity in other systems , is essential for meiotic maturation and oocyte asymmetry. Disrupting CDC42 function by ectopic expression of its GTPase-defective mutants causes both halves of the first meiotic spindle to extend symmetrically toward opposing cortical regions and prevents an asymmetrical division. The elongated spindle has numerous astral-like microtubules, and aPKCzeta, normally associated with the spindle poles, is distributed along its length. Dynactin is displaced from kinetochores, consistently homologous chromosomes do not segregate, and polar body extrusion is prevented. Perturbing the function of aPKCzeta also causes elongation of the meiotic spindle but still permits spindle migration and polar body extrusion. Thus, at least two pathways appear to be downstream of CDC42: one affecting the actin cytoskeleton and required for migration of the meiotic spindle, and a second affecting the spindle microtubules in which aPKCzeta plays a role.     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.