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Regular Article |
a Department of Embryology and Teratology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a specific PKC inhibitor. Our study suggests that exocytosis can be triggered independently either by a [Ca2+]i rise or by PKC.
calcium, fertilization, gamete biology, meiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PKC is a key regulatory enzyme participating in signal transduction pathways that govern cellular responses such as exocytosis. PKC is a family of serine/threonine kinases comprised of three subfamilies depending on their cofactor requirements. The conventional isoforms (cPKC: alpha, beta I, beta II, gamma) require Ca2+ and DAG for maximal activity; the novel isoforms (nPKC: delta, theta, epsilon, mu) are Ca2+ independent but DAG dependent; the atypical isoforms (aPKC: lambda, zeta, iota) require neither Ca2+ nor DAG for their activity. The different cofactor requirements presented by the PKC family probably reflect separate, and maybe even opposing, roles in cellular processes [6].
In all mammalian eggs studied so far, one of the very early cellular events observed after sperm-egg interaction is an increase in intracellular calcium concentration ([Ca2+]i) followed by [Ca2+]i oscillations [5, 7, 8]. Many studies support the role of the [Ca2+]i rise during egg activation as a mediator of CR and of resumption of meiosis. Induction of a single [Ca2+]i rise by exposure to Ca2+ ionophore leads to the triggering of the early as well as the late events of egg activation, whereas utilizing a Ca2+ chelator inhibits both [9–12].
It was shown in various cellular systems that [Ca2+]i elevation and PKC activation are both essential and often act in synergy to elicit a full cellular response [13]. The role of PKC during activation of the mammalian egg is still controversial. Activating PKC in eggs arrested at the second meiotic division (MII) by biologically active phorbol esters has been shown to induce CR, to modify the ZP, and to cause the block to polyspermy [14–16]. In human eggs, PKC activators were demonstrated as possible effectors of the Ca2+ signal, mainly through the ryanidine-sensitive Ca2+ stores [17]. In a recent study, we showed that PKC activation induced only CR without affecting [Ca2+]i levels or cell cycle resumption, whereas the [Ca2+]i rise triggered both aspects of egg activation [16].
Several studies demonstrated the presence of PKC isozymes in eggs and early embryos. PKC alpha, beta I, beta II, gamma, delta, and zeta were identified in Xenopus eggs [18]. Eight PKC isozymes, i.e., cPKC alpha, beta, and gamma; nPKC delta, epsilon, and mu; and aPKC zeta and lambda, were detected in rat eggs [19]. Conventional PKC alpha and gamma, nPKC delta and mu, and aPKC zeta and lambda were detected in unfertilized MII mouse eggs [20, 21] and in embryos from the 2-cell to the 8-cell stage, whereas cPKC beta could not be detected in unfertilized eggs or in early embryos up to the 8-cell stage [21].
Some studies demonstrated that PKC activation leads to CR along with resumption of meiosis [22–26] and second polar body (PBII) extrusion [24, 27]. CR could be accomplished without a change in [Ca2+]i levels or without cell cycle resumption [16, 28]. However, when attempting to interfere with PKC action during fertilization, PKC inhibitors failed to block the fertilization-induced CR [29]. In a previous study, we inhibited PKC activation by staurosporine. The application of 0.1–5 nM staurosporine resulted in an inhibition of CR in a dose-dependent manner (up to 55%). A more significant inhibition of CR could not be achieved because higher concentrations of staurosporine were cytotoxic to the eggs [16].
An important initial step toward understanding the role of PKC in the egg activation process was to study the expression of several PKC isozymes and their capability to be activated during egg parthenogenetic activation or during fertilization. Immunohistochemistry of MII-arrested rat eggs exhibited activation of the cPKC isozymes PKC alpha, beta I, beta II, abd gamma within several minutes of exposure to phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) [19]. PKC translocation in mouse eggs fertilized in vivo was observed about 40 min after sperm penetration (during PBII formation) [26]. Luria et al. [30] showed the activation of PKC alpha and beta I 10–60 min after in vitro insemination of mouse eggs, but they were unable to demonstrate activation of cPKC beta II.
It is not known whether the PKC is stimulated in eggs at early stages of in vivo fertilization. It is also not clear whether the parthenogenetic activators TPA, 1-oleoyl-2-acetylglycerol (OAG), and ionomycin cause CR by the same mechanism as occurs during sperm-induced egg activation. To further study the role of PKC in egg activation and resolve whether Ca2+ and PKC trigger separate pathways to accomplish CR, we undertook three research directions to find out: Are cPKC isoforms activated during in vivo fertilization? Does the pseudosubstrate domain, which can serve as a PKC inhibitor, inhibit CR after parthenogenetic activation? Can additional mechanisms, which may be Ca2+-independent, lead to a complete exocytosis?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MII eggs For induction of ovulation, 23- to 26-day-old immature Wistar-derived female rats were injected with 10 IU human chorionic gonadotropin (hCG; Sigma, St. Louis, MO) 48–54 h after administration of 10 IU of pregnant mares' serum gonadotropin (PMSG; Syncro-part, Sanofi, France). Rats were killed 14 h after the hCG administration. Cumulus-enclosed MII eggs were isolated from the oviducal ampullae into Toyoda HEPES (TH) medium [31] supplemented with 0.4% BSA. Cumulus cells were removed by a brief exposure to 400 IU/ml of highly purified hyaluronidase (H-3631; Sigma) in TH medium.
At all times, the temperature of the medium was kept at 37°C. All manipulations were performed on a binocular warm stage (37°C) placed inside a temperature-controlled hood.
In vivo inseminated eggs Rats were caged with males of proven fertility after the injection of hCG. They were killed 15 h after the hCG administration. Eggs were isolated from the oviducal ampullae at different stages of fertilization. They were classified according to the various stages of fertilization, as previously described for the hamster [32]. These eggs were isolated into TH medium, and their cumulus cells were removed as described for MII eggs. The stages of fertilization were classified as sperm binding (SB), fertilization cone (FC), and the PBII stages 0–15 min, 0–1 h, and 1–3 h after sperm attachment, respectively.
Parthenogenetic Activation
MII-ovulated eggs were parthenogenetically activated by adding three different activators to the incubation medium; all these treatments are capable of inducing a full CR in rat eggs [16].
1. A 3-min incubation in the presence of 2 µM calcium ionophore (ionomycin 407950; Calbiochem, San Diego, CA) followed by an additional 0-, 2-, 7-, or 17-min incubation in fresh medium lacking the activator. A stock solution of ionomycin was prepared as 10 mM in dimethylsulfoxide (DMSO) and kept at 4°C.
2. A 5-min incubation in the presence of 30–50 ng/ml TPA (Sigma) followed by an additional 0-, 5-, or 15-min incubation in fresh medium lacking the activator. A stock solution of TPA was prepared as 1 mg/ml in DMSO and kept at -20°C.
3. A 3-min incubation in the presence of 20 µg/ml OAG (Sigma) followed by an additional 0-, 2-, 5-, or 15-min incubation in fresh medium lacking the activator. A stock solution of OAG was prepared as 1 mg/ml in DMSO and kept at -20°C.
For PKC inhibition, eggs were incubated in the presence of 35 µM myristoylated PKC pseudosubstrate (myrPKC, amino acid 19-27; Biomol, Plymouth, PA) together with each of the different activators at the same concentrations and durations as in the activation experiments. A stock solution of myrPKC was prepared as 1 mM in DMSO and kept at -20°C.
Following activation by the different activators/inhibitor and a further period of incubation, the eggs were fixed and processed as will be described in the next section.
Immunofluorescence Staining and Laser-Scanning Confocal Microscopy
Fixation of eggs Eggs at different stages of development were isolated and fixed for 10 min at room temperature in 3% paraformaldehyde in Dulbecco phosphate-buffered saline (DPBS), supplemented by 0.01% glutaraldehyde, and then washed in a solution of 3% fetal calf serum (FCS; Biological Industries, Beit-Haemek, Israel) in DPBS (DPBS/FCS). Zonae pellucidae were removed postfixation by 0.25% pronase (Sigma) prepared in DPBS/FCS solution, and the ZP-free eggs were washed again in DPBS/FCS.
Detection of CR Fixed eggs were transferred into a blocking solution, i.e., DPBS supplemented with 1% bovine serum albumin (BSA, fraction V; Sigma) and then labeled by lens culinaris aectin (LCA), which binds specifically to cortical granule (CG) content and exudate [33, 34]. Eggs were stained with 5 µg/ml LCA-biotin (Vector, Burlingame, CA), washed, and stained with 1 µg/ml Texas-red streptavidin (Vector).
Permeabilization Zona-free eggs were permeabilized by a 10-min incubation in a solution of 0.05% NP-40 in DPBS/FCS and were then washed in 0.005% NP-40 in DPBS/FCS (blocking solution).
Eggs were incubated for 2 h in the presence of anti-PKC alpha, beta I, or beta II antibodies (1:500 in DPBS/FCS; Santa Cruz Biotechnology, Santa Cruz, CA). Primary antibodies were detected using fluorescent-labeled Cy secondary antibody (1:1000; Jackson Immunoresearch Laboratories, West Grove, PA). Specificity of staining was demonstrated by incubating each primary antibody for 1 h with an excess of the appropriate peptide (Santa Cruz Biotechnology).
Nuclear labeling and developmental stage assessment Eggs were incubated for an additional 10 min with 1 µg/ml of the DNA-specific fluorochrome (Hoechst 33342; Sigma) as a tool of assessing the chromatin stage. Resumption of meiosis was analyzed by monitoring the separation of the chromosomal dyads and the PBII extrusion. The various stages of fertilization were determined by following the sperm and egg chromatin.
Visualization and photography CG exudate, PKC labeling, and DNA staining were visualized and photographed with a Zeiss confocal laser-scanning microscope (CLSM). The Zeiss (Oberkochen, Germany) LSM 410 is equipped with a 25-mW krypton-argon laser, a 10-mW helium-neon laser (488, 543, and 633 maximum lines), and an UV laser (Coherent Inc. Laser Group, Santa Clara, CA). A 40x NA/1.2 planapochromat water immersion lens (Axiovert 135 M; Zeiss) was used for all imaging.
Assessment of PKC translocation For localization of PKC, eggs were scanned using the CLSM through the Z-axis to perform a section at the equatorial plane of the egg. Confocal micrographs of 3–4 eggs from each experimental group were densitometrically analyzed. The stain intensity was measured using the corrected mean density values obtained by the LSM software. PKC translocation was evaluated by calculating the ratio between the PKC signal at the egg membrane and at the cytosol.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunofluorescence Localization of PKC Isozymes During Fertilization
Eggs, before or after sperm penetration (Fig. 1), were labeled by primary antibodies (anti-PKC alpha, beta I, or beta II), LCA, and Hoechst. The ratio between the membrane and cytosol signals was calculated. A relatively uniform distribution of PKC alpha, beta I, and beta II was observed throughout the cytosol of MII-arrested eggs (Fig. 2, A, I, and Q). Upon sperm binding, all three PKC isozymes accumulated at the egg's plasma membrane (Fig. 2, B, J, and R; Fig. 3). Both PKC alpha and beta I presented similar kinetics of translocation to the plasma membrane, demonstrating a small increase in the membrane/cytosol ratio already at the sperm binding (SB) stage (Fig. 2, B and J; Fig. 3). This ratio was further increased until the egg reached the fertilizing cone (FC) stage (Fig. 2, C and K; Fig. 3) and then decreased following the PBII extrusion (Fig. 2, D and L; Fig. 3). No significant difference was found between the kinetics of PKC alpha and PKC beta I, as tested by two-way ANOVA test, whereas PKC beta II was significantly different (P < 0.001). PKC beta II demonstrated a more rapid translocation, the maximum occurring already at the SB stage (Fig. 2R, Fig. 3), followed by a decrease at the FC stage and the PBII extrusion stage (Fig. 2, S and T; Fig. 3). Our results demonstrate activation of these three cPKC isozymes during in vivo fertilization. Activation of PKC beta II was significant (P < 0.005) at the SB stage, whereas that of PKC alpha and beta I became significant (P < 0.005 for alpha; P < 0.001 for beta I) only at the FC stage (one-way ANOVA followed by Tukey multiple comparisons). No CR was observed in MII eggs (Fig. 2, E, M, and W). The CR process was first detected at the SB stage and developed up to the PBII stage (Fig. 2, F–H, N–P, and V–Z).
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PKC Alpha Translocation Following Activation by Various Activators
To study the intracellular signaling pathways leading to the CR, eggs were parthenogenetically activated by the PKC activators TPA or OAG or by ionomycin. The use of immunofluorescence confocal microscopy enabled us to demonstrate activation of cPKC alpha during the parthenogenetic activation by 20 µg/ml OAG or by 50 ng/ml TPA but not by 2 µM ionomycin (Fig. 4). PKC alpha accumulation at the plasma membrane was first detected 5 min after exposure to TPA and reached a maximum at 20 min (Fig. 4, B and C), whereas in eggs activated by OAG, translocation of PKC alpha to the plasma membrane reached a maximum already at 5 min after beginning the activation stimulus (Fig. 4, E and F). No PKC alpha translocation was observed after exposure to ionomycin (Fig. 4, G, H, and I).
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Effect of a PKC Inhibitor on PKC Alpha Translocation and on CR Caused by Various Activators
To resolve the role of PKC in CR, we stimulated CR in the presence of a PKC inhibitor. Eggs were activated by TPA, OAG, or ionomycin and exposed to myrPKC. Immunofluorescence confocal microscopy was used to localize the PKC in the eggs. TPA did not cause resumption of the cell cycle (Fig. 5, A and B); however, it induced PKC alpha translocation (Fig. 5E) and CR (Fig. 5H).
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The presence of both 35 µM myrPKC and 30 ng/ml TPA in the culture medium resulted in an inhibition of PKC alpha translocation (Fig. 5F) and of CR (Fig. 5I).
To evaluate the relationship among [Ca2+]i, CR, and PKC activation, eggs were activated by 2 µM ionomycin (Fig. 6) or 20 µg/ml OAG (Fig. 7) in the presence or absence of 35 µM myrPKC. Under these conditions, CR was not inhibited (ionomycin, Fig. 6I; OAG, Fig. 7b, C). Resumption of the cell cycle occurred similar to that of the control eggs (ionomycin, Fig. 6, A–C; OAG, Fig. 7a, A–C, and b, A). OAG-induced PKC alpha translocation was inhibited by the myrPKC (Fig. 7a, F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A few studies demonstrated the expression of different PKC isozymes and their capability to be activated in eggs. Different PKC isozymes were identified in eggs of some vertebrates [18–21]. Because the CR is regarded as a Ca2+-dependent exocytotic process and because the Ca2+-dependent cPKC alpha and beta II are exocytosis mediators in various cell systems [35, 36], we chose to study the activation of the cPKC. Our results demonstrate, for the first time, activation of the cPKC alpha, beta I, and beta II during in vivo fertilization. Luria et al. [30] showed translocation of cPKC alpha and beta I but not cPKC beta II in mouse egg, 10–60 min after in vitro insemination.
In the current study, immunohistochemistry combined with confocal microscopy revealed a relatively uniform distribution of cPKC alpha, beta I, and beta II throughout the cytosol of MII-arrested rat eggs. All three isozymes examined presented translocation to the plasma membrane as early as the SB stage, when the eggs are still at the MII stage. However, the kinetics of their translocation was not identical: cPKC beta II showed maximum translocation already at the SB stage, while cPKC alpha and beta I were only at the initial stages of translocation and achieved the maximum at the FC stage.
Electrophysiological data demonstrate that the CR in hamster eggs begins as early as 4 sec after sperm binding to the oolemma; however, the dispersal of the content of CGs over the cell surface is relatively slow [37]. The method we used to examine the CR in the current study, namely, visualization of CG content that was dispersed on the egg plasma membrane, allowed us to determine the occurrence of CR already a few minutes after the actual process took place. In an earlier study, PKC translocation to the membrane could be demonstrated only at the stage of the PBII formation (about 40 min postfertilization) [26]. In the present study, translocation of cPKC beta II to the egg membrane was observed in some eggs at the early stage of SB, when CR could not yet be detected (data not shown).
Our results suggest the possibility that cPKC alpha and beta I are involved during the later stages of the egg activation, such as the PBII-formation stage. However, these results do not exclude the possibility of the involvement of cPKC alpha and beta I in the early events of egg activation such as CR because their early translocation from the cytosol to the plasma membrane (already at the SB stage) might be sufficient for inducing the CR. PKC beta I is not a known participant in the exocytotic processes. Since cPKC beta I reached the maximum translocation to the plasma membrane at the FC stage, it might be involved in the later events of egg activation.
We chose to focus on PKC alpha as a representative of the cPKCs because of its abundance [19], its involvement in the exocytosis process [38], and its capability of being activated during parthenogenetic egg activation and fertilization. To evaluate the pathways leading to CR, eggs were parthenogenetically activated by ionomycin or by the PKC activators TPA or OAG. TPA is an activator of PKC, acting similarly to the physiological activator DAG [39]. In the rat egg, TPA induces only CR without triggering [Ca2+]i elevation or resumption of meiosis [16]. OAG, the synthetic DAG, induces both CR [14, 15] and a [Ca2+]i rise in the egg [16]. Ionomycin triggers a [Ca2+]i rise and leads to both CR and resumption of meiosis [16]. Our study demonstrates translocation of cPKC alpha during parthenogenetic activation of the egg by OAG or by TPA but not by ionomycin. PKC alpha translocation caused by OAG was faster than that caused by TPA. Because OAG causes [Ca2+]i elevation while TPA does not and because [Ca2+]i elevation by itself (caused by ionomycin) does not cause PKC alpha translocation, we may deduce that Ca2+ accelerates PKC alpha activation caused by OAG.
The observation that PKC is activated as a result of either parthenogenetic or sperm activation led us to examine the effect of the PKC inhibitor myrPKC on egg activation. The myrPKC is a specific PKC inhibitor, which mimics the effect of the physiological inhibiting subunit that causes PKC autoinhibition. In a previous study, the use of another PKC inhibitor, staurosporine, caused up to 55% inhibition of the CR in rat eggs. A more significant inhibition of the CR could not be achieved because higher concentrations of staurosporine have a cytotoxic effect on the eggs [16]. Our current work reveals that eggs treated with TPA in the presence of myrPKC exhibited inhibition of PKC alpha translocation to the cell membrane in concomitance with CR inhibition. This phenomenon implies that TPA induces CR directly by activating PKC. Jones [28] suggested that high concentrations of TPA cause [Ca2+]i oscillations. The present work supports our previous study, which indicated that there is no [Ca2+]i rise after activation by TPA at a low concentration [16].
An intracellular Ca2+ rise during parthenogenetic activation or fertilization leads to a series of cellular events, the earliest of which is CR. Previous works raised the question as to the mechanism leading to CR after the [Ca2+]i rise [10, 16, 19]. In the current work, PKC alpha translocation was followed in ionomycin-activated eggs. Ionomycin triggered a [Ca2+]i rise in the eggs [16] but did not cause PKC alpha translocation from the cytosol to the egg membrane, indicating that the PKC alpha was not fully activated. This result implies that, under the conditions described, the CR was triggered directly by [Ca2+]i elevation. As described in several biological systems, two conformational changes occur during PKC activation [40]. After the [Ca2+]i rise, Ca2+ binds to cPKC via special Ca2+ binding sites. This interaction causes PKC to undergo a conformational change that leads to the exposure of otherwise hidden sites such as hydrophobic sites [40–42] and probably of the antibody-binding domain. The exposure of hydrophobic sites enhances the enzyme's affinity to the membrane, where it binds to DAG. DAG enables a complete PKC activation including its docking to the cell membrane and an additional conformation change, which causes the release of the pseudosubstrate from the PKC catalytic domain. The catalytic domain is then able to bind to a protein substrate and phosphorylate it [40]. During fertilization, PIP2 is being hydrolyzed to IP3 and DAG [43]. In our study, activation of eggs by ionomycin did not result in translocation of PKC to the membrane. We speculate that this result is due to the fact that DAG, which resides in the egg membrane and serves to dock the PKC, is not produced in ionomycin-induced egg activation. During in vivo fertilization, PKC is activated, as observed by the translocation of the three isozymes studied.
Because PKC is only partially activated by a [Ca2+]i rise, we could not inhibit CR by the use of myrPKC. This implies that [Ca2+]i is able to cause CR, independent of PKC activation. However, we do not rule out the possibility that the [Ca2+]i rise during fertilization acts in synergy with DAG by accelerating PKC activation. The ability of OAG to accelerate PKC activation supports this possibility. Both fertilization and OAG treatment induce a [Ca2+]i rise and PKC activation in the egg. OAG mimics fertilization better than TPA. By applying a PKC inhibitor, we could prevent PKC activation but not the CR, implying that the PKC pathway was bypassed and the CR was triggered by the [Ca2+]i elevation. It should be noted that causing a total block of PKC alpha translocation was more difficult in eggs treated with OAG than with TPA. Even in those eggs that exhibited a total PKC inhibition, the CR was not inhibited. We speculate that the [Ca2+]i rise and activated PKC act in synergy.
We conclude that, in vivo, shortly after sperm binding to the egg plasma membrane, cPKC alpha, beta I, and beta II are activated. In vitro, cPKC can be activated within minutes by TPA [19] or by OAG but not by ionomycin. Although a [Ca2+]i rise by itself does not trigger PKC alpha activation, it can accelerate the activation of PKC alpha that was initially triggered by OAG. Our present study suggests that complete CR can be triggered independently either by a [Ca2+]i rise or by PKC activation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence. FAX: 972 3 6406149; shalgir{at}post.tau.ac.il 
Accepted: February 13, 2002.
Received: November 21, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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