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

International audienceAsymmetric 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

Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCα and PKCγ in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1–3 μM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry

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

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca2+ 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 PLCζ (phospholipase C-ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5-bisphosphate hydrolytic activity of PLCζ 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

Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

We thank Anna-Lena Berg (AstraZeneca, Lund) and Cheryl Scudamore (MRC, Harwell, UK) for histological analysis, Julie Foster (Barts Cancer Institute, London) for CT scans, Johan Swinnen and Frank Claessens (Leuven University, Belgium) for discussion and AR-luciferase reporter plasmids, Florian Guillou (INRA, CNRS, Université de Tours, France) for the AMH-Cre mouse line and Laura Milne (MRC Centre for Reproductive Health, The University of Edinburgh) for technical support. We thank the members of the Cell Signalling group for critical input.International audienceThe organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β 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 p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β 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 p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β 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 p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β 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 p110β and AR have previously been reported to functionally interac

Monitoring Calcium Oscillations in Fertilized Mouse Eggs

International audienceIn 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

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

International audienceAsymmetric 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 requiresMyotonic dystrophy kinase-Related Cdc42-binding Kinase (MRCK), downstream of polarized Cdc42. MRCK inhibitionresulted in spindle rotation defects during anaphase-II, precluding polar body extrusion. Remarkably, disengagement ofsegregated chromatid from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathwayis activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. Thesefindings provide novel insights into the mechanism of myosin II activation in oocytes, and its role in orchestrating asymmetric division and pronucleus centration

MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

Abstract Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In mouse oocytes arrested at metaphase II, eccentric spindle positioning is associated with a chromatin-induced remodeling of the overlying cortex, 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 was demonstrated, the role of ring myosin II, and its mechanism of activation, have remained elusive. Here, we show that ring myosin II activation requires Myotonic dystrophy kinase-Related Cdc42-binding Kinase (MRCK), downstream of polarized Cdc42. During anaphase-II, inhibition of MRCK resulted in spindle rotation defects and a decreased rate of polar body emission. Remarkably, some oocytes eventually achieved spindle rotation by disengaging one cluster of chromatids from the anaphase spindle. We show that the MRCK/myosin II pathway also regulates the flattening of the fertilization cone to initiate male pronucleus centration. These findings provide novel insights into mammalian oocyte polarization and the role of cortical myosin II in orchestrating asymmetric division

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 at meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread microvilli elongation, 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

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

International audienceAsymmetric 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