scholarly journals Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 221-231 ◽  
Author(s):  
Efrat Eliyahu ◽  
Nataly Shtraizent ◽  
Alina Tsaadon ◽  
Ruth Shalgi
Keyword(s):  
Secretory Process ◽  
Egg Activation ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Kinase Substrate ◽  
Kinase C ◽  
Cortical Granule Exocytosis ◽  
Rat Eggs ◽  
Mammalian Eggs ◽  
Pkc Activation

Cortical granule exocytosis (CGE), following egg activation, is a secretory process that blocks polyspermy and enables successful embryonic development. CGE can be triggered independently by either a rise in intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC). The present study investigates the signal transduction pathways leading to CGE through activation of PKC or stimulation of a rise in [Ca2+]i. Using Western blot analysis, co-immunoprecipitation and immunohistochemistry, combined with various inhibitors or activators, we investigated the link between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and CGE. We were able to demonstrate translocation of MARCKS from the plasma membrane to the cortex, in fertilized as well as in parthenogenetically activated eggs. MARCKS phosphorylation was demonstrated upon PKC activation, whereas a PKC inhibitor (myrPKCψ) prevented both MARCKS translocation and CGE in 12-O-tetradecanoyl phorbol-13-acetate (TPA)-activated eggs. We have further shown that upon egg activation the amount of phosphorylated MARCKS (p-MARCKS) and the amount of calmodulin bound to MARCKS were increased. MARCKS translocation in ionomycin activated eggs was also inhibited by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W7). These results complement other studies showing MARCKS requirement for exocytosis and imply that upon fertilization, MARCKS translocation is followed by CGE. These findings present a significant contribution to our understanding of CGE in mammalian eggs in particular, as well as cellular exocytosis in general.

Activation of amphibian oocytes by sperm extracts

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 303-308 ◽  
Author(s):  
F. Bonilla ◽  
M. T. Ajmat ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J. Oterino ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Dna Replication ◽  
Egg Activation ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Granule Exocytosis ◽  
Bufo Arenarum ◽  
Sperm Extract ◽  
Cortical Granule Exocytosis

SummaryIn the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

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.

scholarly journals Strontium-induced rat egg activation

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 467-474 ◽  
Author(s):  
R Tomashov-Matar ◽  
D Tchetchik ◽  
A Eldar ◽  
R Kaplan-Kraicer ◽  
Y Oron ◽  
...  
Keyword(s):  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Egg Activation ◽  
Cortical Granule ◽  
Strontium Chloride ◽  
Quantitative Immunohistochemistry ◽  
Cortical Granule Exocytosis ◽  
Rat Eggs ◽  
Trisphosphate Receptor ◽  
Mouse Eggs

Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca2+]i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca2+]itransient. Strontium chloride (SrCl2) binds to and activates the Ca2+-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca2+]ioscillations. Although SrCl2has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca2+]ioscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl2(2, 4 or 6 mM) on [Ca2+]i, by monitoring [Ca2+]ioscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl2was optimal for inducing the first [Ca2+]itransient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca2+]ioscillations were lower and longer in SrCl2-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl2, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca2+]ioscillations (8, 15 or 60 min in SrCl2respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl2caused a delay in CM. Our results demonstrate that SrCl2is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca2+]ioscillations, CGE and CM.

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