MRCK activates mouse oocyte myosin II for spindle rotation and male pronucleus centration.

Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning triggers cortical polarization, including the build-up of an actin cap surrounded by a ring of activated myosin II. While the role of the actin cap in promoting polar body formation is established, ring myosin II activation mechanisms and functions have remained elusive. Here, we show that ring myosin II activation requires myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), downstream of polarized Cdc42. MRCK inhibition resulted in spindle rotation defects during anaphase II, precluding polar body extrusion. Remarkably, disengagement of segregated chromatids from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathway is activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. These findings provide novel insights into the mechanism of myosin II activation in oocytes and its role in orchestrating asymmetric division and pronucleus centration.

Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

Polo-like kinase 1 promotes Cdc42-induced actin polymerization for asymmetric division in oocytes.

Polo-like kinase I (Plk1) is a highly conserved seronine/threonine kinase essential in meiosis and mitosis for spindle formation and cytokinesis. Here, through temporal application of Plk1 inhibitors, we identify a new role for Plk1 in the establishment of cortical polarity essential for highly asymmetric cell divisions of oocyte meiosis. Application of Plk1 inhibitors in late metaphase I abolishes pPlk1 from spindle poles and prevents the induction of actin polymerization at the cortex through inhibition of local recruitment of Cdc42 and Neuronal Wiskott-Aldrich Syndrome protein (N-WASP). By contrast, an already established polar actin cortex is insensitive to Plk1 inhibitors, but if the polar cortex is first depolymerized, Plk1 inhibitors completely prevent its restoration. Thus, Plk1 is essential for establishment but not maintenance of cortical actin polarity. These findings indicate that Plk1 regulates recruitment of Cdc42 and N-Wasp to coordinate cortical polarity and asymmetric cell division.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition.

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10-/- mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the protein's role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10-/- mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

Cofilin regulates actin network homeostasis and microvilli length in mouse oocytes.

How multiple actin networks coexist in a common cytoplasm while competing for a shared pool of monomers is still an ongoing question. This is exemplified by meiotic maturation in the mouse oocyte, which relies on the dynamic remodeling of distinct cortical and cytoplasmic F-actin networks. Here, we show that the conserved actin-depolymerizing factor cofilin is activated in a switch-like manner upon meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread elongation of microvilli, while cytoplasmic F-actin was depleted, leading to defects in spindle migration and polar body extrusion. In contrast, cofilin inactivation in metaphase II-arrested oocytes resulted in a shutdown of F-actin dynamics, along with a dramatic overgrowth of the polarized actin cap. However, inhibition of the Arp2/3 complex to promote actin cap disassembly elicited ectopic microvilli outgrowth in the polarized cortex. These data establish cofilin as a key player in actin network homeostasis in oocytes and reveal that microvilli can act as a sink for monomers upon disassembly of a competing network.

RhoA- and Cdc42-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes.

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II-arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined.

Monitoring Calcium Oscillations in Fertilized Mouse Eggs.

In mammalian species, including human, fertilization is characterized by the triggering of long-lasting calcium (Ca(2+)) oscillations in the egg cytoplasm. The monitoring of these Ca(2+) oscillations is a valuable technique to demonstrate that fertilization has occurred, to study egg activation events elicited downstream of the Ca(2+) signal, as well as to evaluate sperm quality. This chapter describes our protocol to monitor sperm-induced Ca(2+) oscillations in mouse eggs, using fluorescence microscopy techniques and the Fura-2-AM ratiometric Ca(2+) indicator.

Novel Role for p110ß PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells.

The organismal roles of the ubiquitously expressed class I PI3K isoform p110ß remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110ß, we document that full inactivation of p110ß leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110ß kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110ß results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110ß was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110ß also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110ß inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110ß inactivation. In line with a crucial role for p110ß in SCs, selective inactivation of p110ß in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110ß and AR have previously been reported to functionally interact.

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Cav1.2 channels.

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits ß2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor ß2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation.

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.

Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes.

Asymmetric meiotic divisions in mammalian oocytes rely on the eccentric positioning of the spindle and the remodeling of the overlying cortex, resulting in the formation of small polar bodies. The mechanism of this cortical polarization, exemplified by the formation of a thick F-actin cap, is poorly understood. Cdc42 is a major player in cell polarization in many systems; however, the spatio-temporal dynamics of Cdc42 activation during oocyte meiosis, and its contribution to mammalian oocyte polarization, have remained elusive. In this study, we investigated Cdc42 activation (Cdc42-GTP), dynamics and role during mouse oocyte meiotic divisions. We show that Cdc42-GTP accumulates in restricted cortical regions overlying meiotic chromosomes or chromatids, in a Ran-GTP-dependent manner. This polarized activation of Cdc42 is required for the recruitment of N-WASP and the formation of F-actin-rich protrusions during polar body formation. Cdc42 inhibition in MII oocytes resulted in the release of N-WASP into the cytosol, a loss of the polarized F-actin cap, and a failure to protrude the second polar body. Cdc42 inhibition also resulted in central spindle defects in activated MII oocytes. In contrast, emission of the first polar body during oocyte maturation could occur in the absence of a functional Cdc42/N-WASP pathway. Therefore, Cdc42 is a new protagonist in chromatin-induced cortical polarization in mammalian oocytes, with an essential role in meiosis II completion, through the recruitment and activation of N-WASP, downstream of the chromatin-centered Ran-GTP gradient.

Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes.

Mammalian oocytes are arrested at the prophase of meiosis I during fetal or postnatal development, and the meiosis is resumed by the preovulatory surge of luteinizing hormone. The in vivo functional roles of cyclin-dependent kinases (Cdks) during the resumption of meiosis in mammalian oocytes are largely unknown. Previous studies have shown that deletions of Cdk3, Cdk4 or Cdk6 in mice result in viable animals with normal oocyte maturation, indicating that these Cdks are not essential for the meiotic maturation of oocytes. In addition, conventional knockout of Cdk1 and Cdk2 leads to embryonic lethality and postnatal follicular depletion, respectively, making it impossible to study the functions of Cdk1 and Cdk2 in oocyte meiosis. In this study, we generated conditional knockout mice with oocyte-specific deletions of Cdk1 and Cdk2. We showed that the lack of Cdk1, but not of Cdk2, leads to female infertility due to a failure of the resumption of meiosis in the oocyte. Re-introduction of Cdk1 mRNA into Cdk1-null oocytes largely resumed meiosis. Thus, Cdk1 is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. We also found that Cdk1 maintains the phosphorylation status of protein phosphatase 1 and lamin A/C in oocytes in order for meiosis resumption to occur.

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation.

BACKGROUND INFORMATION: At fertilization in mammalian eggs, the sperm induces a series of Ca(2+) oscillations via the production of inositol 1,4,5-trisphosphate. Increased inositol 1,4,5-trisphosphate production appears to be triggered by a sperm-derived PLCzeta (phospholipase C-zeta) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5-bisphosphate hydrolytic activity of PLCzeta implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization-mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. RESULTS: We investigated PLCzeta- and fertilization-mediated generation of DAG in mouse eggs by monitoring plasma-membrane translocation of a fluorescent DAG-specific reporter. Consistent plasma-membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCzeta, was barely detectable. However, when PLCzeta is overexpressed in eggs, significant plasma-membrane DAG production occurs in concert with a series of unexpected secondary high-frequency Ca(2+) oscillations. We show that these secondary Ca(2+) oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca(2+) oscillations appears to involve Ca(2+) influx and the loading of thapsigargin-sensitive Ca(2+) stores. CONCLUSIONS: Our results suggest that overproduction of DAG in PLCzeta-injected eggs can lead to PKC-mediated Ca(2+) influx and subsequent overloading of Ca(2+) stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca(2+) homoeostasis via excessive Ca(2+) influx.

Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes.

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca(2+) oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca(2+)] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca(2+) oscillations in the cytosol that are transduced into mitochondrial Ca(2+) oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP](mito) and [ATP](cyto). We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca(2+)](cyto) and [ATP](cyto) during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.

Constitutive PtdIns(3,4,5)P3 synthesis promotes the development and survival of early mammalian embryos.

Mammalian preimplantation embryos develop in the oviduct as individual entities, and can develop and survive in vitro, in defined culture media lacking exogenous growth factors or serum. Therefore, early embryos must generate intrinsic signals that promote their development and survival. In other cells, activation of class I phosphoinositide 3-kinase (PI3K) is a universal mechanism to promote cell proliferation and survival. Here, we examined whether PI3K is intrinsically activated during preimplantation development. Using GFP-tagged pleckstrin homology domains to monitor PtdIns(3,4,5)P(3) synthesis, we show that PI3K is constitutively activated in mouse preimplantation embryos. E-cadherin ligation promotes PtdIns(3,4,5)P(3) synthesis at sites of blastomere adhesion at all cleavage stages. In addition, in culture conditions that promote autocrine signalling, a second pool of PtdIns(3,4,5)P(3) is generated in the apical membrane of early stage blastomeres. We show that constitutive PtdIns(3,4,5)P(3) synthesis is necessary for optimal development to blastocyst and to prevent large-scale apoptosis at the time of cavitation.

Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes.

Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. We investigated the role of Rac in oocyte meiosis by using a fluorescent reporter for Rac-GTP. We find that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation caused a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex.

Imaging phosphoinositide dynamics using GFP-tagged protein domains.

Phosphoinositides are important regulators of cellular homoeostasis and numerous signal-transduction pathways. One of their major features is their ability to recruit signalling proteins to membranes by direct interaction with phosphoinositide-binding modules. The distribution and dynamics of membrane phosphoinositides are therefore major determinants in the spatiotemporal control of cell signalling and membrane trafficking. However, standard biochemical approaches cannot reveal the dynamics of phosphoinositides at the single-cell level. A major technical advance has been the development of genetically encoded fluorescent phosphoinositide probes on the basis of the phosphoinositide-binding domains found in signalling proteins, such as the PH (pleckstrin homology) domain. This review describes the diverse fluorescent phosphoinositide probes available for imaging specific phosphoinositide species and how their use has improved the understanding of phosphoinositide signalling at the single-cell level.

PKC signaling at fertilization in mammalian eggs.

Protein kinase C (PKC) has been proposed to regulate major egg activation events during mammalian fertilization. Most of the evidence supporting this assumption has first been obtained using pharmacological activation and inhibition of the kinase, while egg activation was assessed by checking for exocytosis of the cortical granules, extrusion of the second polar body and formation of pronuclei. However, results have been inconclusive and sometimes contradictory regarding the exact role of PKC in regulating egg activation events. The PKC family is composed of various isotypes, which differ in their modular structures and regulatory properties. Hence the need to re-examine the roles of egg PKCs more specifically. Mammalian eggs express many PKC isotypes, the roles of which have been investigated using immunodetection, isotype-specific inhibition and, more recently, live imaging of fluorescent chimaeras. Here, I review the recent development of PKC research in mammalian fertilization and the evidence for a specific role for certain PKC isotypes in fertilization-induced egg activation.

PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane.

Phosphatidylinositol 3-kinase (PI3K) has been shown to enhance native voltage-dependent calcium channel (Ca(v)) currents both in myocytes and in neurons; however, the mechanism(s) responsible for this regulation were not known. Here we show that PI3K promotes the translocation of GFP-tagged Ca(v) channels to the plasma membrane in both COS-7 cells and neurons. We show that the effect of PI3K is mediated by Akt/PKB and specifically requires Ca(v)beta(2) subunits. The mutations S574A and S574E in Ca(v)beta(2a) prevented and mimicked, respectively, the effect of PI3K/Akt-PKB, indicating that phosphorylation of Ser574 on Ca(v)beta(2a) is necessary and sufficient to promote Ca(v) channel trafficking.

Ca2+ signalling and cortical re-organisation during the transition from meiosis to mitosis in mammalian oocytes.

In mammals, the mature ovulated egg is arrested in metaphase II of the first meiotic division. The signal that triggers the transition from meiosis to mitosis is provided by the fertilising sperm and takes the form of a series of Ca(2+) oscillations. The pattern of Ca(2+) oscillations is imposed by maternal control mechanisms that ensure Ca(2+) transients occur during M-phase of meiosis II and during the first mitotic division. The transition from meiosis to mitosis involves a major re-organisation. The unfertilised egg is polarised with the meiotic spindle located in the cortex of the animal pole and clusters of endoplasmic reticulum in the vegetal hemisphere. By the time of the first mitotic division some 20h later the spindle has formed in the centre of the embryo and is surrounded by endoplasmic reticulum. These changes in organisation have implications for the inheritance of ER in meiotic and mitotic cell divisions and may reflect different roles and requirements for Ca(2+) in meiosis and mitosis.