The involvement of protein kinase C and actin filaments in cortical granule exocytosis in the rat.
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.
Eliyahu
E
E
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
Tsaadon
A
A
Shtraizent
N
N
Shalgi
R
R
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D004789:Enzyme Activation; D005260:Female; D005455:Fluorescent Antibody Technique; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008677:Metaphase; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism*; D009866:Oogenesis / Q000502:physiology*; D010312:Parthenogenesis; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007476:Ionophores; D007527:Isoenzymes; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate