Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse eggs

1997 ◽  
Vol 46 (2) ◽  
pp. 216-226 ◽  
Author(s):  
Tom Ducibella ◽  
Leah LeFevre
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Kinase C ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs

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

1990 ◽  
Vol 1 (3) ◽  
pp. 315-326 ◽  
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.

scholarly journals The involvement of protein kinase C and actin filaments in cortical granule exocytosis in the rat

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 161-170 ◽  
Author(s):  
E Eliyahu ◽  
A Tsaadon ◽  
N Shtraizent ◽  
R Shalgi
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cortical Granule ◽  
Fluorescence Signal ◽  
Filamentous Actin ◽  
Granule Exocytosis ◽  
Kinase C ◽  
Exact Function ◽  
Cortical Granule Exocytosis

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.

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

1989 ◽  
Vol 108 (3) ◽  
pp. 885-892 ◽  
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.

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs

Development ◽  
1991 ◽  
Vol 112 (2) ◽  
pp. 461-470 ◽  
Author(s):  
N. Grandin ◽  
M. Charbonneau
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Ph ◽  
Phorbol Esters ◽  
Intracellular Free Calcium ◽  
Egg Activation ◽  
Cortical Granule ◽  
Free Calcium ◽  
Kinase C

Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca2+]i). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885–892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca2+ transient. We report here that PKC activators do not induce the intracellular Ca2+ transient, or the activation-associated increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597–604, 1990).

290. Calmodulin-dependent protein kinase II, and not protein kinase C, transduces the Ca2+ signal at fertilization

2005 ◽  
Vol 17 (9) ◽  
pp. 122
Author(s):  
K. T. Jones ◽  
M. Levasseur ◽  
H. Chang ◽  
S. Madgwick
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Polar Body ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Polar Body Extrusion ◽  
Mouse Eggs ◽  
Constitutively Active

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell cycle resumption. Here we used a combination of pharmacological inhibitors and constitutively-active cRNA constructs of PKCα and CamKII microinjected into mouse eggs, to show that it is CamKII and not PKC that is the sufficient trigger for cell cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on meiotic resumption but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to meiotic resumption, the effects of constitutively-active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced meiotic resumption but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell cycle resumption through stimulation of CamKII.

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