Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

Mammalian sperm–egg fusion results in cortical granule exocytosis (CGE) and resumption of meiosis. Studies of various exocytotic cells suggest that filamentous actin (F-actin) blocks exocytosis by excluding secretory vesicles from the plasma membrane. However, the exact function of these microfilaments, in mammalian egg CGE, is still elusive. In the present study we investigated the role of actin in the process of CGE, and the possible interaction between actin and protein kinase C (PKC), by using coimmunoprecipitation, immunohistochemistry and confocal microscopy. We identified an interaction between actin and the PKC alpha isoenzyme in non-activated metaphase II (MII) eggs and in eggs activated by phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). F-actin was evenly distributed throughout the egg’s cytosol with a marked concentration at the cortex and at the plasma membrane. A decrease in the fluorescence signal of F-actin, which represents its depolymerization/reorganization, was detected upon fertilization and upon parthenogenetic activation. Exposing the eggs to drugs that cause either polymerization or depolymerization of actin (jasplakinolide (JAS) and cytochalasin D (CD) respectively) did not induce or prevent CGE. However, CD, but not JAS, followed by a low dose of TPA doubled the percentage of eggs undergoing complete CGE, as compared with TPA alone. We further demonstrated that myristoylated alanin-rich C kinase substrate (MARCKS), a protein known to cross-link F-actin in other cell types, is expressed in rat eggs and is colocalized with actin. In view of our results, we suggest that the cytoskeletal cortex is not a mere physical barrier that blocks CGE, but rather a dynamic network that can be maneuvered towards allowing CGE by activated actin-associated proteins and/or by activated PKC.

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Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs.

The Journal of Cell Biology ◽

10.1083/jcb.108.3.885 ◽

1989 ◽

Vol 108 (3) ◽

pp. 885-892 ◽

Cited By ~ 79

Author(s):

W M Bement ◽

D G Capco

Keyword(s):

Protein Kinase C ◽

Retinoic Acid ◽

Protein Kinase ◽

Xenopus Laevis ◽

Follicle Cells ◽

Cortical Granule ◽

Cleavage Furrow ◽

Granule Exocytosis ◽

Kinase C ◽

Cortical Granule Exocytosis

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.

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Translocation of the Classic Protein Kinase C Isoforms in Porcine Oocytes: Implications of Protein Kinase C Involvement in the Regulation of Nuclear Activity and Cortical Granule Exocytosis

Experimental Cell Research ◽

10.1006/excr.2002.5547 ◽

2002 ◽

Vol 277 (2) ◽

pp. 183-191 ◽

Cited By ~ 31

Author(s):

Heng-Yu Fan ◽

Chao Tong ◽

Man-Yu Li ◽

Li Lian ◽

Da-Yuan Chen ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cortical Granule ◽

Nuclear Activity ◽

Granule Exocytosis ◽

Kinase C ◽

Cortical Granule Exocytosis ◽

Protein Kinase C Isoforms

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Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Cell Regulation ◽

10.1091/mbc.1.3.315 ◽

1990 ◽

Vol 1 (3) ◽

pp. 315-326 ◽

Cited By ~ 51

Author(s):

W M Bement ◽

D G Capco

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Mitotic Cell ◽

Cortical Granule ◽

Mitotic Cell Cycle ◽

Cleavage Furrow ◽

Kinase C ◽

Cortical Granule Exocytosis

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.

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Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes

PLoS ONE ◽

10.1371/journal.pone.0135679 ◽

2015 ◽

Vol 10 (8) ◽

pp. e0135679 ◽

Cited By ~ 14

Author(s):

Matilde de Paola ◽

Oscar Daniel Bello ◽

Marcela Alejandra Michaut

Keyword(s):

Cortical Granule ◽

Granule Exocytosis ◽

Mouse Oocytes ◽

Sensitive Factor ◽

Cortical Granule Exocytosis

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Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

Molecular Biology of the Cell ◽

10.1091/mbc.e11-07-0614 ◽

2011 ◽

Vol 22 (24) ◽

pp. 4908-4917 ◽

Cited By ~ 26

Author(s):

Deepti Gadi ◽

Alice Wagenknecht-Wiesner ◽

David Holowka ◽

Barbara Baird

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Time Course ◽

Fluorescent Protein ◽

Immunoglobulin E ◽

Dependent Manner ◽

Effector Domain ◽

Granule Exocytosis ◽

Kinase C

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+concentrations and oscillatory association of PKCβ–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

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