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.

Are Src family kinases involved in cell cycle resumption in rat eggs?

The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca2+ transients. The pathways, within the egg, leading to the intracellular Ca2+ release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca2+ release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity (PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca2+ elevation, is an SFK-dependent process, while CGE, triggered by either Ca2+ elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm-egg fusion site downstream of the Ca2+ release remains unclear.

Signal transduction pathways in activation of the mammalian egg.

The mature egg arrested at its second metaphase is a rather quiet cell, and possesses a pre-set developmental program. A sequence of biochemical events that leads to the rapid embryonic mitotic divisions initiates at fertilization when the spermatozoon overcomes this second cell cycle arrest. These events induce a transient rise in intracellular Ca2+ concentration ([Ca2+]i) that leads to the cortical granule exocytosis (CGE) and resumption of meiosis. Various treatments can induce parthenogenetic activation as manifested by the extrusion of the second polar body or CGE. Similar to somatic cells, recent studies in mammalian eggs suggest that signal transduction pathways mediate egg activation. The initial increase in ([Ca2+]i) appears to be critical for egg activation. Other messengers such as protein kinase C and protein tyrosine kinases, were suggested as possible inducers of some aspects of egg activation.

PKC in eggs and embryos.

After a spermatozoon enters the egg, rapid processes such as the cortical granules exocytosis (CGE) occur. Other, later processes are observed as well, and they include resumption of the second meiotic division, second polar body (PBII) extrusion and rearrangement of the DNA inside the pronuclei (PN). The above mentioned biochemical processes involve signal transduction pathways that are well known in other cell systems, and require mediation of second messengers like Ca2+ and diacylglycerol (DAG) which are protein kinase C (PKC) activators. The present review, based upon recently published studies, raises the possibility of PKC involvement in fertilization and in early developmental stages of the mammalian embryo.

Profile of protein kinase C isozymes and their possible role in mammalian egg activation.

Western blot analysis was used to investigate protein kinase C (PKC) profile of rat eggs. The presence of eight PKC isozymes was demonstrated: conventional PKC alpha, beta and gamma; novel PKC delta, epsilon and mu; atypical PKC zeta and lambda. PKC alpha was detected by RT-PCR as well. PKC translocation from the cytosol to the plasma membrane served as a marker for enzyme activation. Immunofluorescence confocal microscopy demonstrated a relatively uniform distribution of PKC alpha, betaI, and betaII throughout the cytosol of metaphase II arrested eggs. PKC accumulation at the plasma membrane was detected 5 min after exposure to 12-O-tetradecanoyl phorbol-13-acetate and increased with time, thus demonstrating activation of these PKCs.

Structure and activity of the translocated c-myc in mouse plasmacytoma XRPC-24.

In the mouse plasmacytoma XRPC-24 both c-mos and c-myc are rearranged. We cloned the rearranged c-myc and found that it was translocated to the immunoglobulin C alpha locus. The breakpoint is at the end of exon 1 in c-myc and approximately 0.5 kb upstream from exon 1 of C alpha. The cloned translocated c-myc linked to a strong transcriptional promoter can efficiently transform rat embryo fibroblasts when co-transfected with the activated Ha-ras. The transformed cells are tumorigenic in syngeneic rats.