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Protein kinase C initially inhibits the induction of meiotic cell division in xenopus oocytes
Cellular Signalling Vol. 4, No. 4, pp. 393-403, 1992. Printed in Great Britain.
PROTEIN
KINASE
MEIOTIC
0898--6568/92 $5.00 + 0.00 ~ 1992 Pergamon Press Ltd
C INITIALLY CELL
INHIBITS
DIVISION
THE
IN XENOPUS
INDUCTION
OF
OOCYTES
BRADLEY J. STITH,* M A R C L. GOALSTON~ and ALLAN J. K I R K W O O D Department of Biology, Universityof Colorado at Denver, Denver, C O 80217-3364, U.S.A.
(Received 20 December 1991; and accepted 10 February 1992) Abstract--We have used one activator and two inhibitors of protein kinase C (PKC) to examine the role of this enzyme in the induction of meiotic cell division. At 1 U/ml, phosphatidylcholine-specific phospholipase C increases DAG, alters intracellular pH and inhibits the induction of meiosis by insulin or progesterone. However, when added about 1.6 h after progesterone, the enzyme speeds the induction of cell division. Microinjection of inhibitor peptide (19-36) of PKC has little effect on progesterone action but stimulates the induction of meiosis by insulin. When the inhibitor peptide is injected about 2 h after insulin addition, the peptide inhibits. A second PKC inhibitor, staurosporine, decreases PKC-dependent intracellular pH and /n vitro oocyte PKC activity. At similar concentrations, staurosporine stimulates insulin or progesterone action, but, when added after about 2 h, the drug inhibits induction by insulin. We conclude that PKC is initially inhibitory to the induction of meiotic cell division but then may become synergistic.
Key words: Insulin, progesterone, staurosporine, phospholipase C, cell division, inhibitor peptide, insulin-like growth factor 1. INTRODUCTION A NEW pathway to meiosis in Xenopus oocytes was suggested when it was found that a phorbol ester can induce meiotic cell division [1]. Phorbol esters mimic endogenous diacylglycerol (DAG) to stimulate protein kinase C (PKC). Other groups have since reported phorbol ester-induced meiosis [2-6]. As the phorbol ester may be acting through a mechanism not involving PKC, the enzyme was purified from rat brain and microinjected into oocytes [1, 4]. Both laboratories report that this isozymic mixture of PKC was not sufficient to induce meiosis, although it did potentiate insulin- and ras-induced meiosis and at least *Author to whom correspondenceshould be addressed at: University of Colorado at Denver, Biology(171), P.O. Box 173364, Denver, CO 80217-3364, U.S.A. Abbreviations: DAG--sn- 1,2-diacylglycerol; 1.0 GVBD50---time required for 50% of the oocytes to enter prophase; IP3---inositol 1,4,5-trisphosphate; PC-PLC--phosphatidylcholine-specifi¢ phospholipase C; PI-PLC--phosphatidylinositol-spe¢ifi¢ phospholipase C; PlP2--phosphatidylinositol 4,5-bisphosphate; PKC--Caand DAG-dependent protein kinase C. 393
one proliferative event (increased $6 phosphorylation). More recent evidence [7] lends support for the PKC pathway in that insulin, insulin-like growth factor 1, and progesterone induce a biphasic increase in D A G levels before cells enter prophase. The size of the first peak of D A G corresponded to the ability of the hormone to induce meiotic cell division. Furthermore, the early D A G peak occurs just before hormone-induced proliferative events such as an increase in intracellular pH [1]. The second peak of D A G is temporally associated with development of the cortical endoplasmic reticulum [8], and nuclear migration [3]. Similar concentrations o f insulin (about 50nM) or IGF-1 (0.5 nM) induce 50% of the oocytes to undergo meiosis and a half-maximal late D A G increase. Ras p21 microinjection presumably induces meiosis through an increase in D A G and PKC activation not through inositol 1,4,5trisphosphate (IP3) or cAMP changes ([9-11]; although see [12]). In addition, ras p21 can mimic some actions o f phorbol esters on oocytes [13]. PKC induction o f cell division
B.J. STITHet al.
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may be a widespread phenomenon as it is required for the G2-to-M transition in the yeast S. cerevisiae [14] and over-expression of the P K C B gene promotes proliferation in rat fibroblasts [15]. In opposition to a synergistic role for P K C in meiotic cell division, two reports suggest that P K C is actually inhibitory to the induction of meiosis [6, 16]. The work presented here expands upon the apparent ability of P K C to both inhibit and stimulate the induction of meiotic cell division. Through use of two inhibitors and a presumptive activator of PKC, we find that P K C is inhibitory to the induction o f meiosis. However, 2-3 h before entry into prophase, P K C may become synergistic to the induction of meiotic cell division. MATERIALS AND METHODS
Xenopus females were obtained from Xenopus One (Ann Arbor, MI) and maintained on a diet of cubed beef heart. Three to seven days before use, the female frogs were injected with 35 IU of pregnant mare's serum gonadotropin (Sigma) to decrease the time required for meiotic cell division, to increase the percentage of responding cells, and to increase the synchrony of cell division [17]. Oocytes were obtained by manual dissection of ovarian fragments and no spontaneous maturation was noted after 24 h. Highly pure phosphatidylcholine-specific phospholipasc C (PC-PLC; 2000 U/mg; no lipolytic contaminates) was obtained from Boehringer Mannheim (Indianapolis, IN). Phosphoinositide-specific phospholipase C (PI-PLC; 1500 U/ml) was obtained from Dr Martin Low (Columbia University). Staurosporine from Streptomyces sp. (Bochringer Mannheim) was solubilized in dimethyl sulphoxide (DMSO), and kept protected from light at 4°C. Inhibitor peptide (19-36; Gibeo BRL; Grand Island, NY) stock solution was 100/~M in 20raM Tris (pH 7.5). PKC activity was assayed as describexi in [1]. For studies on the time required to enter meiosis, groups of 20 oocytcs were placed in 3 ml of O-R2 (83raM NaCl, 0.SmM CaCl2, 1 mM MgCl2, and l0 mM HEPES; pH 7.9). When phospholipasc C was used, 0.2% BSA (insulin-free, Reheis Chemical Co.) was added to the external medium. Microinjection of 30nl of a 100#M inhibitor peptide (19-36 PKC) solution into oocytes (a dilution of about 15-fold) was accomplished with the use of a PV830 PicoPump (World Precision Instruments, Sarasota, FL).
Progesterone (5#M) or insulin (1 #M) was added and entry into prophase was followed by noting the occurrence of a white spot on the animal pole. This spot represents a prophase event called nuclear or germinal vesicle breakdown (GVBD). Oocytes were fixed in 5% TCA and dissected to confirm the lack of a nucleus. The rate of GVBD was obtained by recording the number of oocytes that have undergone division and expressing this as a percentage vs the time of the recording. A method to standardize the period before entry into cell division was used since the induction period varied from about 4 to 7 h due to the use of different growth factors and cells from different frogs. The time of hormone addition was set to zero, and the time required for 50% of the cells to divide (GVBD50) was transformed to 1.0. Typically, the period of induction was about 4 h for progesterone and about 5-6 h for insulin, so that 0.4 GVBD50 would be 1.6-2.4 h. Analysis of DAG mass was by use of DAG kinase [7]. Intracellular pH in oocytes was measured as described in Ref. [17]. Calcium levels in oocytes were measured by fluorescence with a Quantex system after microinjection of 30-50 nl of 10 mM Fura-2. Data are presented in figures as the average +S.E.M., and, in the text, as the average +S.D. Differences between experimental and control groups were examined by the pooled Student's t-test.
RESULTS
Presumptive protein kinase C activator inhibits hormone-induced meiosis if added early, but stimulates if added after about 0.4 G VBDSO Addition of 1 U/ml of PC-PLC increases D A G levels very rapidly (Fig. 1A; similar results were obtained in two other experiments). We then attempted to follow P K C translocation to the membrane with PC-PLC addition. However, five different monoclonal antibodies against mammalian P K C did not bind Xenopus P K C (collaboration with K. Leach, Upjohn Laboratories). Finally, as the sodium-hydrogen exchanger is believed to be regulated by P K C (phorbol ester will increase intracellular p H in oocytes; [1]), we used intracellular p H as a measure o f in vivo P K C activity. Since PC-PLC increases intraceUular p H when added at a concentration between 0.5 and 1 U/ml (Fig. I B), we suggest that these concentrations of PC-PLC were sufficient to activate P K C in the oocyte.
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FIG. 1. Phosphatidylcholine-specific phospholipase C addition to Xenopus oocytes causes an increase in DAG levels and increases intracellular pH. (A) Addition of ! U/mi of PC-PLC induced a 10-fold increase in DAG mass followed by a decline to a plateau (about a six-fold increase). The first point is the control group, the second is at 5 s after enzyme addition, the third is at 30 s and the fourth is at I min. All experimental groups have significantly higher DAG levels (~ < 0.05). Each point represents three determinations. (B) PC-PLC concentrations greater than 0.5 U/ml increase intracellular pH (a PKC-dependent event). A weak acid (14C-dimethyloxazolidine dione) that equilibrates across the oocyte membrane in a pH-dependent manner and various concentrations of PC-PLC were incubated with cells for 1 h and intracelluiar pH determined [1, 17, 61]. Each point represents four determinations and each determination was made on five oocytes. Asterisks denote significance at ~ < 0.0001.
Similar concentrations of PC-PLC (about 0.5U/ml) inhibited progesterone- or insulininduced meiotic cell division (four experiments). These results are represented in Fig. 2 which shows the concentration of external enzyme required to inhibit insulin- or progesteroneinduced meiosis. In five experiments, extracel-
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FIG. 2. Phosphatidylcholine-specific phospholipase C inhibits both insulin- and progesterone-induced meiosis in a dose-dependent manner. The ordinate represents the percentage of cells that have entered prophase. (A) PC-PLC inhibition of 2/~M insulininduced meiosis. Insulin alone (open square), and with PC-PLC addition (0.1 U/ml, closed circle; 0.5 U/ml, open triangle; 1 U/ml, closed triangle) are shown. (B) PC-PLC inhibition of 5 #M progesteroneinduced meiosis. Progesterone alone (open circle), and with various PC-PLC concentrations (open triangle, 0.005 U/ml; closed square, 0.1 U/ml; open inverted triangle, 0.3 U/ml; closed inverted triangle, 0.6 U/ml; open diamond, 1 U/ml; closed diamond, 3 U/ml) are shown. lular addition of I U/ml PC-PLC never induced meiosis by itself. Since similar concentrations of PC-PLC are required to inhibit meiosis and to increase intracellular pH, one would also expect that comparable concentrations of PC-PLC are required to increase D A G levels. We note that PC-PLC at 0.1 and 0 , 5 U / m l increased D A G levels by 18 and 30%, respectively (Fig. 3). In an attempt to determine the period of P K C inhibition, PC-PLC was added at various times after hormone addition and the effect on
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the meiotic induction time was recorded. Inhibition of progesterone-induced meiosis was actually reversed when the enzyme was added after about 0.4 GVBD50 (Fig. 4A). Summarizing the data on the late addition of the enzyme, PC-PLC addition after 0.4 GVBD50 resulted in a significant acceleration of progesterone-induced meiosis [approximately 50 min faster; 80.3 + 10.8% (S.D.) of the control groups; n = 4] earlier. Thus, P K C may be inhibitory to the initial induction of cell division by the steroid. However, P K C may act in a positive manner from about 0.4 GVBD50 (1.6 h after progesterone addition) to a b o u t 0.9 GVBD50 (until about the time when 10-20% of the cells have entered prophase). Addition of PC-PLC with insulin was strongly inhibitory, but, when added after about 0.5 GVBD50, the enzyme was much less inhibitory (Fig. 4B). These data show that insulin induction of cell division is much more sensitive to PC-PLC inhibition than that induced by progesterone since the enzyme never stimulates the proliferative action o f insulin. Maximal inhibition of insulin action was obtained when PC-PLC was added 1 h after the peptide hormone (0.16 GVBD50) whereas maximal inhibition o f progesterone-induced meiosis was at time zero. Another difference between the response of the two hormones is
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Fro. 4. Phosphatidylcholine-specific phospholipase C addition suggests that PKC is inhibitory up to about 0.4 GVBD50, but stimulates progesterone-induced meiosis and is less inhibitory toward insulin induction after this early period. (A) Action of PC-PLC on progesterone induction of meiosis. The ordinate is the time required for meiosis (relative to control group; divide the time required for white spot appearance in the experimental group by the time required for the control group) of a group of 15-20 ooeytes. The abscissa is the time of addition of 1 U/mi of PC-PLC. Five micromolar progesterone was used to induce meiosis and these data represent four experiments with ooeytes from four different frogs. (B) Action of PC-PLC on insulin action. One representative experiment out of four is shown: insulin alone (open circle), simultaneous addition of insulin and PC-PLC (ckr~d circle) and PC-PLC addition 1 h (open triangle; at 0.16 GVBD50), 3h (closed triangle; at 0.5 GVBDS0), 4 h (open square; at 0.66 GVBD50) and 5 h (closed square; at 0.82 GVBD50) after insulin addition. Note that early addition of PC-PLC (before 0.5 GVBD50) after insulin addition resulted in maximal inhibition. that PC-PLC inhibited very late in insulininduced meiosis (cf. with the lack of an effect after 0.9 GVBD50 with progesterone; Fig. 4A). Addition of the enzyme when half the cells
Protein kinase C and meiosis showed an insulin-induced white spot (that denotes entry into M phase) slowed attainment of 100% GVBD from 45 min (control cells) to 5.5 h later (Fig. 4B). As a control, equivalent or higher levels of activity of another phospholipase C (PIspecific) were added to cells and this enzyme was unable to affect insulin- or progesteroneinduced meiosis or D A G levels (four experiments). This is evidence that phosphoinositides are not present in the outer membrane leaflet and that addition of a similar protein is not sufficient for these effects. As an additional control, 1 U/ml of PC-PLC was unable to induce a large increase in calcium levels when added to oocytes. To demonstrate that the calcium-dependent fluorescent system was capable of measuring increases in intracellular calcium, 1 #M ionomycin was found to increase oocyte calcium levels within 1 min to levels greater than 1 #M.
Use of protein kinase C inhibitors Inhibitor peptide (PKC amino acid residues 19-36) binds to the active site of PKC to inhibit the enzyme. We chose a stock concentration of peptide so that, after microinjection into oocytes, the final calculated concentration of inhibitor peptide in the cell was 6.7/~M. This value is in the range 5-10/~M which inhibits PKC in cell extracts or in vivo [18]. Microinjection of inhibitor peptide alone did not induce meiosis after 21-24h (n = 8; oocytes from eight different frogs). Microinjection of the inhibitor peptide just before 5 #M progesterone addition did not alter the rate of meiosis when compared with the rate obtained with buffer-injected control cells [4.96 _ 1.07 h (S.D.) for GVBD50 in control cells vs 4.87 _ 1.1 h (S.D.) for inhibitor peptide injected; n = 8; the range for 50% GVBD was from 3.1 to 6.9 h]. In three of these cases, there was a trend toward stimulation (inhibitor peptide groups matured about 0.2, 0.15 and 0.4 h faster than control values o f 4.45, 4.6 and 5.0 h, respectively). This non-significant trend for stimulation was followed by a period where CELLS 4:4-E
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FIG. 5. Microinjection of inhibitor peptide into Xenopus oocytes stimulates and later inhibits insulin induction of meiotic cell division. Inhibitor peptide (100/~M in 20mM Tris, pH7.5) was injected just before and 1, 2, 3, 4 and 5 h after insulin addition to groups of 20 oocytes. Based on an intracellular free volume of about 0.5/zl and an injection volume of 33 nl, the calculated final concentration of peptide was 6.7/~M. Control injections with buffer alone were just before insulin addition and 4 h later, and peptide injected experimental groups were compared to the appropriate control group. The second control injection was always performed since oocytes from some frogs matured more slowly if injected at 4 h after insulin. The ordinate is the time for 50% of the peptide-injected cells to begin meiosis (i.e. white spot appearance) minus the time for 50% of the control cells to enter meiosis [5.1 _+ 0.9 h (S.D.)]. Negative numbers represent a stimulation and positive numbers an inhibition of the induction of meiosis. The abscissa is the time of injection of peptid¢; 0 is just before insulin addition, whereas other values represent injection of peptide some 1, 2, 3, 4 and 5 h after insulin addition. An asterisk represents values significantly different from 0 (~ < 0.05; six experiments with oocytes from six different frogs).
there was a trend toward slight slowing of the induction of meiosis by progesterone (data not shown). Thus, the inhibitor peptide had little effect on progesterone action. With insulin, inhibitor peptide injection just before hormone addition accelerated the rate of meiotic cell division by about 40 min [0.63 + 0.41 h (S.D.), n = 6; Fig. 5] over bufferinjected controls. Injection of the inhibitor peptide at 4 h after insulin addition inhibited induction o f meiosis by about 40 min. Note that the cross-over from stimulation to inhibition is about 2 h (0.4 GVBD50) after insulin addition. These data suggest that PKC initially inhibits
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FIG. 6. Staurosporine inhibits Xenopus PKC and stimulates meiosis at concentrations above 0.1/aM. (A) Various concentrations of staurosporine were added to partially pure PKC [2] and the control samples received equivalent amounts of DMSO. After a 20rain incubation, PKC activity was measured (see Materials and Methods). Basal activity represents five determinations, whereas treatment groups are the average and S.E. of three values. Stars or asterisks represent a significant difference (= < 0.05). (B) Similar concentrations of staurosporine reduce PKC-dependent intracellular pH. Oocytes were incubated with various concentrations of staurosporine for I h then l U/ml PC-PLC and 14Cdimethyloxazolidine dione were added. After a second hour for radioisotope equilibration across the plasma membrane, the intracellular pH was determined. Each point represents at least four groups of
insulin induction of meiosis but, after about 0.4 GVBD50, is synergistic. We then examined the effect of another PKC inhibitor, staurosporine, on the ability of progesterone or insulin to induce meiotic cell division. First, we found that staurosporine inhibits Xenopus PKC activity after the enzyme was partially purified from oocytes by ionexchange resin [2]. The PKC activity eluted off in a sharp peak of activity at an ionic strength reported by Laurent et al. [2], was activated by phorbol ester, and peak activity fractions had greater amounts of a protein at 80,000 mol. wt. Staurosporine concentrations greater than 100nM were needed to inhibit Xenopus PKC (Fig. 6A). We examined whether staurosporine reduced intracellular pH as a measure of the drug's effect o n / n vivo PKC. After preincubation in various concentrations of staurosporine, 1 U/ml PC-PLC was added to the oocytes and intracellular pH determined. Staurosporine concentrations between 0.1 and 0.5/zM were required to reduce stimulated levels of intracellular pH (Fig. 6B) (possibly due to membrane damage or short life, inhibitor peptide did not alter the membrane distribution of the weak acid and our estimate of pH). Although purified PKC is more susceptible, there are six reports that micromolar levels of staurosporine are required for PKC inhibition in cellular extracts or to prevent cell proliferation (see [19], and Ref. [20] for a review). Similar concentrations of staurosporine stimulate progesterone-induced meiosis (Fig. 6C). Based on these dose-response data, 1 #M staurosporine was chosen as a minimal concentration that stimulates meiosis but would be less likely to inhibit enzymes other than PKC. Addition of staurosporine with insulin or up to about 3h after insulin (0.4-0.5 GVBD50) stimulates insulin (Fig. 7A). However, when the
five oocytes. (C) Progesterone (5 #M) induction of meiosis is accelerated by addition of 1/aM staurosporine (data represent three experiments). Control values (closed triangle), 0.1/aM staurosporine (open circle), and !/aM staurosporine (closed circle) are shown.
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potentiates hormone-induced DAG increases; thus, the stimulation of meiosis by staurosporine may reflect overproduction of DAG and stimulation of enzymes that depend on DAG. Various concentrations of the drug were added to groups of 10 ooeytes and DAG levels measured. After a 2 h incubation period, 0.1, 1 and 5 #M staurosporine did not increase DAG levels (two experiments; four determinations at each concentration). In addition, the drug had no effect on progesterone-induced early increase in DAG levels (data not shown).
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FIG. 7. Staurosporine data suggest that PKC is inhibitory early although insulin action may require PKC activity after about 0.5 GVBD50. (A) Early addition of 1/zM staurosporine stimulates but late addition inhibits insulin (2 #M) induction of meiosis. Results with control group (open circle), and groups with staurosporine addition at I h (closed circle), 2 h (open triangle), 3 h (closed triangle; 0.4 GVBD50), 4h (open square; 0.53 GVBD50), and 5h (closed square) after insulin addition are shown. (B) Addition of staurosporine always stimulates progesterone induction of meiosis. Control values (open circle), 1 #M staurosporine addition at 1 h (open triangle), 1.5 h (closed triangle) and 2 h (open square) after progesterone are shown. Insulin and progesterone data represent three experiments each.
drug was added after about 0.5 GVBD50, it inhibited insulin-induced meiosis. Staurosporine addition with progesterone stimulates progesterone-induced meiosis and maximal stimulation is achieved after about 0.3 GVBD50 (about 1.5h after progesterone addition; Fig. 7B). The use of inhibitors is plagued by the problem of non-specificity [21]. For example, Bishop et al. [22] have found that staurosporine
We conclude that PKC inhibits initiation of the processes leading to entry into prophase. Two PKC inhibitors stimulate and one activator inhibits (a total of six treatments with insulin and progesterone) the induction of meiosis when used before about 0.4 GVBD50. Note that this time point is 1.6-2.4h after hormone addition and that prophase begins at 4--6 h. These results agree with earlier reports that PKC inhibits meiotic cell division [6, 16]. We also suggest that PKC promotes the induction of meiosis after about 0.4 GVBD50. Other reports suggest this duality in that phorbol esters will initially enhance or inhibit a response and, when added at later times, produce the opposite effect [23-26]. Protein kinase C inhibition of cell division
PKC may be involved in checkpoints which review commitment to cell division [27]. As growth factors must be present for a period of hours before induction of cell division, this PKC inhibition may be responsible. The inhibitory mechanism may involve a decrease in G protein or phospholipase C activity and a reduction in agonist-stimulated phosphoinositide turnover [22, 28-30]. However, the breakdown of polyphosphoinositide 4,5bisphosphate (PIP2) and calcium release may not be required for growth factor-induced proliferation in many cells including oocytes [1, 11, 29-35].
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A second possible mechanism of PKC inhibition of meiosis could be related to phorbol ester stimulation of adenylate cyclase [28, 36]. PKC could increase cAMP to inhibit proliferation since, in the oocyte, high cAMP levels are inhibitory (see Ref. [37]). However, I/~M phorbol ester has little or no effect upon cAMP levels in Xenopus oocytes [38]. Phorbol ester inhibited meiosis in mouse oocytes yet was unable to prevent the initial decrease in cAMP [39]. These studies suggest that the PKC inhibition of meiotic induction occurs at a point distal to the decrease in cAMP-dependent protein kinase A activity.
Late PKC activity may be predominantly synergistic We also propose that PKC becomes synergistic to the induction of meiotic cell division after about 0.4 GVBD50. At this time (a) the presumptive PKC activator (PC-PLC) stimulates progesterone-induced meiosis, (b) PC-PLC is less inhibitory to insulin-induced meiosis, (c) staurosporine inhibits insulin-induced meiosis, (d) inhibitor peptide injection inhibits insulin action. PKC may be involved in late events that take place from about 0.4 GVBD50 until white spot appearance since DAG again increases over this period and peaks at 0.85 GVBD50 for insulin, progesterone, or insulin-like growth factor 1 [7] (see also Introduction). The change in PKC action may be about 0.4 GVBD50 since at this point (a) staurosporine's effect on insulin switches from stimulation to inhibition, (b) staurosporine reaches maximal stimulation of progesterone, (c) PC-PLC's effect on progesterone switches from inhibition to slight stimulation, and (d) inhibitor peptide stimulation switches to inhibition of insulin action at this point. Thus, three late treatments and the trend shown by inhibitor peptide on progesterone action support the idea of late PKC synergism. However, two late treatments do not: (1) late addition of staurosporine does not inhibit progesterone action, and (2) late addition of PC-PLC does not stimulate insulin action.
These findings could be due to an effect of PCPLC or staurosporine on a pathway not involving PKC. The ability of PC-PLC to stimulate late progesterone but not insulin action may be related to the ability of PKC to phosphorylate and inhibit the insulin and IGF1 receptors [40--43]. At this time, there are no demonstrated effects of PKC on the progesterone receptor in oocytes.
Use of PKC inhibitors and activators Inhibitor peptide is relatively specific to PKC as 2115-fold higher amounts are required to inhibit cAMP-dependent protein kinase A [18]. As opposed to sphingosine [6, 16], this PKC inhibitor alone did not induce meiosis. Staurosporine was also used due to its ability to inhibit PKC [44] and it produced results similar to the inhibitor peptide. As evidence that the drug acts through PKC inhibition, staurosporine concentrations required to decrease intracellular pH were similar to those that were required to effect induction of meiosis. Surprisingly, this PKC inhibitor has been found to be a tumour promoter [45]. Based on the work presented here, this turnout promotion may be due to its ability to reverse the PKC inhibition of the initiation of cell division. In an attempt to avoid the use of phorbol esters and to provide data that can be compared to that obtained by inhibitors, we used PC-PLC to increase DAG and stimulate PKC. Due to the inaccessibility of some phospholipids such as PIP2 to externally added phospholipase [46] and the specific nature of PC-PLC, addition of highly pure PC-PLC to oocytes should only produce effects through increased DAG. In agreement with this belief is our finding that PC-PLC did not cause an increase in intracellular calcium that would represent breakdown of PIP2 to IP3 or general membrane disruption. Finally, PC-PLC may be acting through DAG and PKC as similar concentrations of the enzyme increase DAG, intracellular pH (a PKC-del~ndent process) and affect the induction of meiotic cell division. Further evidence that PC-PLC acts through
Protein kinase C and meiosis PKC activation is found i n the literature as addition of 0.5 U/ml phospholipase C mimics phorbol ester action on rat follicle-enclosed oocytes [47] or hepatocytes [48]. Apparent contradiction: P K C induction and inhibition o f meiosis
Treatments that increase D A G levels (ras p21 microinjection [10]; D A G kinase inhibitor [49]; insulin, IGF-1 or progesterone addition [7]) or addition of a phorbol ester [1] induce meiosis. However, this report demonstrates an initial PKC inhibition of meiosis. This apparent contradiction of PKC action is reflected in the literature. Bornslaeger et al. [39] have shown that phorbol esters inhibited meiosis in mouse oocytes, but Aberdam and Dekel [47] found that phorbol esters stimulated meiosis in follicle-enclosed rat oocytes (see Ref. [50] for a review). Phorbol esters induce meiosis in Spisula yet inhibit 5-hydroxtryptamine-induced meiosis in this cell [51]. In at least three other cell systems, phorbol esters induce proliferation alone yet antagonize growth factor action in the same cell [52]. Phorbol esters induce proliferation in fibroblasts and epithelial cells, act as comitogens in lymphocytes, yet inhibit mitogenesis in muscle and some epithelial cells (see Ref. [53] for a review). Phorbol esters inhibit proliferation of undifferentiated carcinoma cells and stimulate proliferation of differentiated derivatives of these cells [54-56]. Differences in phorbol ester action could be due to the ability of the phorbol esters to stimulate and then down-regulate PKC (i.e. phorbol esters may induce meiosis by actually depleting the oocyte of PKC). This may explain the inability of injected PKC to induce meiosis [1, 4]. One may also suggest that the apparent discrepancy between PKC induction and inhibition is related to the early PKC inhibition and a late PKC stimulation of cell division reported here. Phorbol esters may selectively induce the PKC synergistic pathway whereas PC-PLC and staurosporine affect the inhibitory pathway. As Stith et al. [7] have suggested that the two hormone-induced D A G peaks occur in
401
different locations within the cell, inhibition and stimulation may be due to PKC action in different locations. Different isozymes of PKC may be responsible for the inhibition or stimulation since there are two isozymes of PKC in Xenopus oocytes [57]. Protein kinase C in the induction o f meiotic cell division
Based on the above discussion, a refinement of the model for PKC involvement in the induction of meiotic cell division can be proposed. Insulin acts through the IGF-1 receptor [58] and through ras p21 [59, 60] to increase D A G levels [7, 11]. Progesterone also induces an increase in D A G levels. The resulting activation of PKC induces various proliferative events that may not be required for cell division (such as the increase in intracellular pH [1]) but the overall effect of PKC at this time is inhibition. The period of PKC inhibition is relieved at about 2 h after hormone addition and synergistic PKC pathways predominate. Independent PKC inhibitory and stimulatory paths may be due to the presence of an active enzyme in different subcellular locations or to different PKC isozymes. Acknowledgements--Thanks to CHRISJAYNESand SALLYSILVAfor assistance. This work was supported in part by NSF grant DCB-8916703.
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9. Birchmeier C., Broek D. and Wigler M. (1985) Cell 43, 615-621. 10. Lacal J. C., de la Pena P., Moscat J., GarciaBarreno P., Anderson P. and Aaronson S. (1987) Science 238, 533-536. 11. Lacal J. C. (1990) Melee. cell. Biol. 10, 333-340. 12. Sadler S. E. and Mailer J. L. (1989) J. biol. Chem. 264, 856-861. 13. Johnson A. D., Cork R. J., Williams M. A., Robinson K. R. and Smith L. D. (1990) Cell Regul. 1, 543-554. 14. Levin D. E., Field F. O., Kunisawa R., Bishop J. M. and Thorner J. (1990) Cell 62, 213-224. 15. Hoshina S., Ueffing M. and Weinstein I. B. (1990) J. cell. Physiol. 145, 262-267. 16. Schorderet-Slatkine S. and Urner F. (1986) Tokai J. exp. clin. Med. 11,453-462. 17. Stith B. J. and MaUer J. L. (1984) Devl Biol. 102, 79-89. 18. House C. and Kemp B. E. (1987) Science 238, 1726-1728. 19. Meyer T., Regenass U., Fabbro D., Alteri E., Rosel J., Muller M., Caravatti G. and Matter A. (1989) Int. J. Cancer 43, 851-856. 20. Standaert M. L., Buckley D. J., Ishizuka T., Hoffman J. M., Cooper D. R., Pellet R. J. and Farese R. V. (1990) Metab. clin. Exp. 39, 1170-1179. 21. Smith C. D., Glickman J. F. and Chang K. J. (1988) Biochem. biophys. Res. Commun. 156, 1250-1256. 22. Bishop W. R., August J., Petrin J. M. and Pai J.-K. (1990) Biochem. J. 269, 465--473. 23. Sagi-Eisenberg R., Lieman H. and Pecht I. (1985) Nature 313, 59-60. 24. Wright S. D. and Meyer B. C. (1986) J. Immun. 136, 1759-1764. 25. Zavoico G. B., Halenda S. P., Sha'afi R. I. and Feinstein M. B. (1985) Proc. natn. Acad. Sci. U.S.A. 82, 3859-3862. 26. Degen J. L., Estensen R. D., Nagamine Y. and Reich E. (1985) J. biol. Chem. 260, 12,426-12,433. 27. Hartwell L. H. and Weinert T. A. (1989) Science 246, 629-634. 28. Katada T., Gilman A. G., Watanabe Y., Bauer S. and Jakobs K. H. (1985) Eur. J. Biochem. 151, 431-437. 29. L'Allemain G., Paris S., Magnaldo I. and Pouyssegur J. (1986) J. cell. Physiol. 129, 167-174. 30. Mond J. J., Balapure A., Feurerstein N., June C. H., Brunswick M., Lindsberg M. L. and Witherspoon K. (1990) J. Immun. 144, 451--455. 31. McNeil P. L., McKenna M. P. and Taylor D. L. (1985) J. Cell Biol. 101, 372-379. 32. Pessin M. S., Baldassare J. J. and Rabcn D. M. (1990) J. biol. Chem. 265, 7959-7966.
33. Hill T. D., Dean N. M., Mordan L. J., Lau A. F., Kanemitsu M. Y. and Boynton A. L. (1990) Science 248, 1660-1663. 34. Margolis B., Ziibcrstein A., Francks C., Felder S., Kremer S., Ullrich A. R., Rhee S. G., Skorecki K. and Schlessinger J. (1990) Science 248, 607-610. 35. Nishizawa N., Okano Y., Chatani Y., Amano F., Tanaka E., Nomoto H., Nozawa Y. and Kohno M. (1990) Cell Regul. 1, 747-761 36. Abou-Samra A.-B., Harwood J. P., Manganiello V. C., Cart K. J. and Aguilera G. (1987) J. biol. Chem. 262, 1129-1136. 37. Mailer J. L. (1990) Biochemistry 29, 3157-3166. 38. Greenfield L. J., Hackett J. T. and Linden J. (1990) Am. J. Physiol. 259, C792--C800. 39. Bornslaeger E. A., Poueymirou W. T., Mattei P. and Schultz R. M. (1986) Expl. Cell Res. 165, 507-517. 40. Grunberger G. and Gorden P. (1982) ~4m. J. Physiol. 243, E319-E324. 41. Jacobs S., Sahyoun N. E., Saltiel A. R. and Cuatrecasas P. (1983) Prec. nam. Acad. Sci. U.S.A. 80, 6211-6213. 42. Takayama S., White M. F., Lauris V. and Kahn C. R. (1984) Prec. natn. Acad. Sci. U.S.A. 81, 7797-7801. 43. Haring H., Kirseh D., Obermaier B., Ermel B. and Machicao F. (1986) J. biol. Chem. 261, 3869-3875. 44. Tamaoki T., Nomoto H., Takahashi I., Kate Y., Morimoto M. and Tomita F. (1986) Biochem. biophys. Res. Commun. 135, 397--402. 45. Sake T., Taubcr A. I., Jeng A. Y., Yuspa S. H. and Blumberg P. M. (1988) Cancer Res. 48, 4646-4650. 46. Op den Kamp J. A. F. (1979) A. Rev. Biochem. 48, 47-71. 47. Aberdam E. and Dekel N. (1985) Biochem. biophys. Res. Commun. 132, 570-574. 48. Blackmore P. F., Strickland W.G., Bocekino S. B. and Exton J. H. (1986) Biochem. J. 237, 235-242. 49. Wasserman W. J., Freedman A. B. and LaBella J. J. (1988) J. Cell Biol. 107, 818a. 50. Eckberg W. R. (1988) Biol. Bull. 174, 95-108. 51. Krantic S., Dub¢ F., Quirion R. and Guerrier P. (1991) Devl Biol. 146, 491-498. 52. Sauro M. D. and Zorn N. E. (1991) J. cell. Physiol. 148, 133-138. 53. Whiteley B., Cass¢! D., Zhuang Y.-X. and Glaser L. (1984) J. Cell Biol. 99, 1162-1166. 54. Snoek G. T., Mummery C. L., Van Den Brink C. E., Van Der Saag P. T. and deLaat S. W. (1986) Devl Biol. 115, 282-292. 55. Sahyoun N., Wolf M., Bcsterman J., Hsieh T.-S., Sander M., I.xVine H. III, Change K.-J. and Cuatrecasas P. (1986) Prec. natn. Acad. Sci. U.S.A. 83, 1603-1607.
Protein kinas¢ C and meiosis 56. Darbon J.-M., Valette A. and Bayard F. (1986) Biochem. Pharm. 35, 2683-2686. 57. Chen K.-h., Peng Z.-g., Lavu S. and Kung H.-f. (1988-89) Sec. Mess. Phosphoprot. 12, 251-260. 58. Mailer J. L. and Kooritz J. (1981) Devl Biol. 85, 309-316.
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59. Deshpande A. K. and Kung H.-F. (1987) Molec. cell. Biol. 7, 1285-1288. 60. Kern L. J., Siebel C. W., McCormick F. and Roth R. A. (1987) Science 236, 840-843. 61. Stith B. J. and MaUer J. L. (1985) Devl Biol. 107, 460-469.
PROTEIN
KINASE
MEIOTIC
0898--6568/92 $5.00 + 0.00 ~ 1992 Pergamon Press Ltd
C INITIALLY CELL
INHIBITS
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INDUCTION
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BRADLEY J. STITH,* M A R C L. GOALSTON~ and ALLAN J. K I R K W O O D Department of Biology, Universityof Colorado at Denver, Denver, C O 80217-3364, U.S.A.
(Received 20 December 1991; and accepted 10 February 1992) Abstract--We have used one activator and two inhibitors of protein kinase C (PKC) to examine the role of this enzyme in the induction of meiotic cell division. At 1 U/ml, phosphatidylcholine-specific phospholipase C increases DAG, alters intracellular pH and inhibits the induction of meiosis by insulin or progesterone. However, when added about 1.6 h after progesterone, the enzyme speeds the induction of cell division. Microinjection of inhibitor peptide (19-36) of PKC has little effect on progesterone action but stimulates the induction of meiosis by insulin. When the inhibitor peptide is injected about 2 h after insulin addition, the peptide inhibits. A second PKC inhibitor, staurosporine, decreases PKC-dependent intracellular pH and /n vitro oocyte PKC activity. At similar concentrations, staurosporine stimulates insulin or progesterone action, but, when added after about 2 h, the drug inhibits induction by insulin. We conclude that PKC is initially inhibitory to the induction of meiotic cell division but then may become synergistic.
Key words: Insulin, progesterone, staurosporine, phospholipase C, cell division, inhibitor peptide, insulin-like growth factor 1. INTRODUCTION A NEW pathway to meiosis in Xenopus oocytes was suggested when it was found that a phorbol ester can induce meiotic cell division [1]. Phorbol esters mimic endogenous diacylglycerol (DAG) to stimulate protein kinase C (PKC). Other groups have since reported phorbol ester-induced meiosis [2-6]. As the phorbol ester may be acting through a mechanism not involving PKC, the enzyme was purified from rat brain and microinjected into oocytes [1, 4]. Both laboratories report that this isozymic mixture of PKC was not sufficient to induce meiosis, although it did potentiate insulin- and ras-induced meiosis and at least *Author to whom correspondenceshould be addressed at: University of Colorado at Denver, Biology(171), P.O. Box 173364, Denver, CO 80217-3364, U.S.A. Abbreviations: DAG--sn- 1,2-diacylglycerol; 1.0 GVBD50---time required for 50% of the oocytes to enter prophase; IP3---inositol 1,4,5-trisphosphate; PC-PLC--phosphatidylcholine-specifi¢ phospholipase C; PI-PLC--phosphatidylinositol-spe¢ifi¢ phospholipase C; PlP2--phosphatidylinositol 4,5-bisphosphate; PKC--Caand DAG-dependent protein kinase C. 393
one proliferative event (increased $6 phosphorylation). More recent evidence [7] lends support for the PKC pathway in that insulin, insulin-like growth factor 1, and progesterone induce a biphasic increase in D A G levels before cells enter prophase. The size of the first peak of D A G corresponded to the ability of the hormone to induce meiotic cell division. Furthermore, the early D A G peak occurs just before hormone-induced proliferative events such as an increase in intracellular pH [1]. The second peak of D A G is temporally associated with development of the cortical endoplasmic reticulum [8], and nuclear migration [3]. Similar concentrations o f insulin (about 50nM) or IGF-1 (0.5 nM) induce 50% of the oocytes to undergo meiosis and a half-maximal late D A G increase. Ras p21 microinjection presumably induces meiosis through an increase in D A G and PKC activation not through inositol 1,4,5trisphosphate (IP3) or cAMP changes ([9-11]; although see [12]). In addition, ras p21 can mimic some actions o f phorbol esters on oocytes [13]. PKC induction o f cell division
B.J. STITHet al.
394
may be a widespread phenomenon as it is required for the G2-to-M transition in the yeast S. cerevisiae [14] and over-expression of the P K C B gene promotes proliferation in rat fibroblasts [15]. In opposition to a synergistic role for P K C in meiotic cell division, two reports suggest that P K C is actually inhibitory to the induction of meiosis [6, 16]. The work presented here expands upon the apparent ability of P K C to both inhibit and stimulate the induction of meiotic cell division. Through use of two inhibitors and a presumptive activator of PKC, we find that P K C is inhibitory to the induction o f meiosis. However, 2-3 h before entry into prophase, P K C may become synergistic to the induction of meiotic cell division. MATERIALS AND METHODS
Xenopus females were obtained from Xenopus One (Ann Arbor, MI) and maintained on a diet of cubed beef heart. Three to seven days before use, the female frogs were injected with 35 IU of pregnant mare's serum gonadotropin (Sigma) to decrease the time required for meiotic cell division, to increase the percentage of responding cells, and to increase the synchrony of cell division [17]. Oocytes were obtained by manual dissection of ovarian fragments and no spontaneous maturation was noted after 24 h. Highly pure phosphatidylcholine-specific phospholipasc C (PC-PLC; 2000 U/mg; no lipolytic contaminates) was obtained from Boehringer Mannheim (Indianapolis, IN). Phosphoinositide-specific phospholipase C (PI-PLC; 1500 U/ml) was obtained from Dr Martin Low (Columbia University). Staurosporine from Streptomyces sp. (Bochringer Mannheim) was solubilized in dimethyl sulphoxide (DMSO), and kept protected from light at 4°C. Inhibitor peptide (19-36; Gibeo BRL; Grand Island, NY) stock solution was 100/~M in 20raM Tris (pH 7.5). PKC activity was assayed as describexi in [1]. For studies on the time required to enter meiosis, groups of 20 oocytcs were placed in 3 ml of O-R2 (83raM NaCl, 0.SmM CaCl2, 1 mM MgCl2, and l0 mM HEPES; pH 7.9). When phospholipasc C was used, 0.2% BSA (insulin-free, Reheis Chemical Co.) was added to the external medium. Microinjection of 30nl of a 100#M inhibitor peptide (19-36 PKC) solution into oocytes (a dilution of about 15-fold) was accomplished with the use of a PV830 PicoPump (World Precision Instruments, Sarasota, FL).
Progesterone (5#M) or insulin (1 #M) was added and entry into prophase was followed by noting the occurrence of a white spot on the animal pole. This spot represents a prophase event called nuclear or germinal vesicle breakdown (GVBD). Oocytes were fixed in 5% TCA and dissected to confirm the lack of a nucleus. The rate of GVBD was obtained by recording the number of oocytes that have undergone division and expressing this as a percentage vs the time of the recording. A method to standardize the period before entry into cell division was used since the induction period varied from about 4 to 7 h due to the use of different growth factors and cells from different frogs. The time of hormone addition was set to zero, and the time required for 50% of the cells to divide (GVBD50) was transformed to 1.0. Typically, the period of induction was about 4 h for progesterone and about 5-6 h for insulin, so that 0.4 GVBD50 would be 1.6-2.4 h. Analysis of DAG mass was by use of DAG kinase [7]. Intracellular pH in oocytes was measured as described in Ref. [17]. Calcium levels in oocytes were measured by fluorescence with a Quantex system after microinjection of 30-50 nl of 10 mM Fura-2. Data are presented in figures as the average +S.E.M., and, in the text, as the average +S.D. Differences between experimental and control groups were examined by the pooled Student's t-test.
RESULTS
Presumptive protein kinase C activator inhibits hormone-induced meiosis if added early, but stimulates if added after about 0.4 G VBDSO Addition of 1 U/ml of PC-PLC increases D A G levels very rapidly (Fig. 1A; similar results were obtained in two other experiments). We then attempted to follow P K C translocation to the membrane with PC-PLC addition. However, five different monoclonal antibodies against mammalian P K C did not bind Xenopus P K C (collaboration with K. Leach, Upjohn Laboratories). Finally, as the sodium-hydrogen exchanger is believed to be regulated by P K C (phorbol ester will increase intracellular p H in oocytes; [1]), we used intracellular p H as a measure o f in vivo P K C activity. Since PC-PLC increases intraceUular p H when added at a concentration between 0.5 and 1 U/ml (Fig. I B), we suggest that these concentrations of PC-PLC were sufficient to activate P K C in the oocyte.
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FIG. 1. Phosphatidylcholine-specific phospholipase C addition to Xenopus oocytes causes an increase in DAG levels and increases intracellular pH. (A) Addition of ! U/mi of PC-PLC induced a 10-fold increase in DAG mass followed by a decline to a plateau (about a six-fold increase). The first point is the control group, the second is at 5 s after enzyme addition, the third is at 30 s and the fourth is at I min. All experimental groups have significantly higher DAG levels (~ < 0.05). Each point represents three determinations. (B) PC-PLC concentrations greater than 0.5 U/ml increase intracellular pH (a PKC-dependent event). A weak acid (14C-dimethyloxazolidine dione) that equilibrates across the oocyte membrane in a pH-dependent manner and various concentrations of PC-PLC were incubated with cells for 1 h and intracelluiar pH determined [1, 17, 61]. Each point represents four determinations and each determination was made on five oocytes. Asterisks denote significance at ~ < 0.0001.
Similar concentrations of PC-PLC (about 0.5U/ml) inhibited progesterone- or insulininduced meiotic cell division (four experiments). These results are represented in Fig. 2 which shows the concentration of external enzyme required to inhibit insulin- or progesteroneinduced meiosis. In five experiments, extracel-
18 TIME AFTER PROGESTERONE (HRSI
FIG. 2. Phosphatidylcholine-specific phospholipase C inhibits both insulin- and progesterone-induced meiosis in a dose-dependent manner. The ordinate represents the percentage of cells that have entered prophase. (A) PC-PLC inhibition of 2/~M insulininduced meiosis. Insulin alone (open square), and with PC-PLC addition (0.1 U/ml, closed circle; 0.5 U/ml, open triangle; 1 U/ml, closed triangle) are shown. (B) PC-PLC inhibition of 5 #M progesteroneinduced meiosis. Progesterone alone (open circle), and with various PC-PLC concentrations (open triangle, 0.005 U/ml; closed square, 0.1 U/ml; open inverted triangle, 0.3 U/ml; closed inverted triangle, 0.6 U/ml; open diamond, 1 U/ml; closed diamond, 3 U/ml) are shown. lular addition of I U/ml PC-PLC never induced meiosis by itself. Since similar concentrations of PC-PLC are required to inhibit meiosis and to increase intracellular pH, one would also expect that comparable concentrations of PC-PLC are required to increase D A G levels. We note that PC-PLC at 0.1 and 0 , 5 U / m l increased D A G levels by 18 and 30%, respectively (Fig. 3). In an attempt to determine the period of P K C inhibition, PC-PLC was added at various times after hormone addition and the effect on
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the meiotic induction time was recorded. Inhibition of progesterone-induced meiosis was actually reversed when the enzyme was added after about 0.4 GVBD50 (Fig. 4A). Summarizing the data on the late addition of the enzyme, PC-PLC addition after 0.4 GVBD50 resulted in a significant acceleration of progesterone-induced meiosis [approximately 50 min faster; 80.3 + 10.8% (S.D.) of the control groups; n = 4] earlier. Thus, P K C may be inhibitory to the initial induction of cell division by the steroid. However, P K C may act in a positive manner from about 0.4 GVBD50 (1.6 h after progesterone addition) to a b o u t 0.9 GVBD50 (until about the time when 10-20% of the cells have entered prophase). Addition of PC-PLC with insulin was strongly inhibitory, but, when added after about 0.5 GVBD50, the enzyme was much less inhibitory (Fig. 4B). These data show that insulin induction of cell division is much more sensitive to PC-PLC inhibition than that induced by progesterone since the enzyme never stimulates the proliferative action o f insulin. Maximal inhibition of insulin action was obtained when PC-PLC was added 1 h after the peptide hormone (0.16 GVBD50) whereas maximal inhibition o f progesterone-induced meiosis was at time zero. Another difference between the response of the two hormones is
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Fro. 4. Phosphatidylcholine-specific phospholipase C addition suggests that PKC is inhibitory up to about 0.4 GVBD50, but stimulates progesterone-induced meiosis and is less inhibitory toward insulin induction after this early period. (A) Action of PC-PLC on progesterone induction of meiosis. The ordinate is the time required for meiosis (relative to control group; divide the time required for white spot appearance in the experimental group by the time required for the control group) of a group of 15-20 ooeytes. The abscissa is the time of addition of 1 U/mi of PC-PLC. Five micromolar progesterone was used to induce meiosis and these data represent four experiments with ooeytes from four different frogs. (B) Action of PC-PLC on insulin action. One representative experiment out of four is shown: insulin alone (open circle), simultaneous addition of insulin and PC-PLC (ckr~d circle) and PC-PLC addition 1 h (open triangle; at 0.16 GVBD50), 3h (closed triangle; at 0.5 GVBDS0), 4 h (open square; at 0.66 GVBD50) and 5 h (closed square; at 0.82 GVBD50) after insulin addition. Note that early addition of PC-PLC (before 0.5 GVBD50) after insulin addition resulted in maximal inhibition. that PC-PLC inhibited very late in insulininduced meiosis (cf. with the lack of an effect after 0.9 GVBD50 with progesterone; Fig. 4A). Addition of the enzyme when half the cells
Protein kinase C and meiosis showed an insulin-induced white spot (that denotes entry into M phase) slowed attainment of 100% GVBD from 45 min (control cells) to 5.5 h later (Fig. 4B). As a control, equivalent or higher levels of activity of another phospholipase C (PIspecific) were added to cells and this enzyme was unable to affect insulin- or progesteroneinduced meiosis or D A G levels (four experiments). This is evidence that phosphoinositides are not present in the outer membrane leaflet and that addition of a similar protein is not sufficient for these effects. As an additional control, 1 U/ml of PC-PLC was unable to induce a large increase in calcium levels when added to oocytes. To demonstrate that the calcium-dependent fluorescent system was capable of measuring increases in intracellular calcium, 1 #M ionomycin was found to increase oocyte calcium levels within 1 min to levels greater than 1 #M.
Use of protein kinase C inhibitors Inhibitor peptide (PKC amino acid residues 19-36) binds to the active site of PKC to inhibit the enzyme. We chose a stock concentration of peptide so that, after microinjection into oocytes, the final calculated concentration of inhibitor peptide in the cell was 6.7/~M. This value is in the range 5-10/~M which inhibits PKC in cell extracts or in vivo [18]. Microinjection of inhibitor peptide alone did not induce meiosis after 21-24h (n = 8; oocytes from eight different frogs). Microinjection of the inhibitor peptide just before 5 #M progesterone addition did not alter the rate of meiosis when compared with the rate obtained with buffer-injected control cells [4.96 _ 1.07 h (S.D.) for GVBD50 in control cells vs 4.87 _ 1.1 h (S.D.) for inhibitor peptide injected; n = 8; the range for 50% GVBD was from 3.1 to 6.9 h]. In three of these cases, there was a trend toward stimulation (inhibitor peptide groups matured about 0.2, 0.15 and 0.4 h faster than control values o f 4.45, 4.6 and 5.0 h, respectively). This non-significant trend for stimulation was followed by a period where CELLS 4:4-E
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FIG. 5. Microinjection of inhibitor peptide into Xenopus oocytes stimulates and later inhibits insulin induction of meiotic cell division. Inhibitor peptide (100/~M in 20mM Tris, pH7.5) was injected just before and 1, 2, 3, 4 and 5 h after insulin addition to groups of 20 oocytes. Based on an intracellular free volume of about 0.5/zl and an injection volume of 33 nl, the calculated final concentration of peptide was 6.7/~M. Control injections with buffer alone were just before insulin addition and 4 h later, and peptide injected experimental groups were compared to the appropriate control group. The second control injection was always performed since oocytes from some frogs matured more slowly if injected at 4 h after insulin. The ordinate is the time for 50% of the peptide-injected cells to begin meiosis (i.e. white spot appearance) minus the time for 50% of the control cells to enter meiosis [5.1 _+ 0.9 h (S.D.)]. Negative numbers represent a stimulation and positive numbers an inhibition of the induction of meiosis. The abscissa is the time of injection of peptid¢; 0 is just before insulin addition, whereas other values represent injection of peptide some 1, 2, 3, 4 and 5 h after insulin addition. An asterisk represents values significantly different from 0 (~ < 0.05; six experiments with oocytes from six different frogs).
there was a trend toward slight slowing of the induction of meiosis by progesterone (data not shown). Thus, the inhibitor peptide had little effect on progesterone action. With insulin, inhibitor peptide injection just before hormone addition accelerated the rate of meiotic cell division by about 40 min [0.63 + 0.41 h (S.D.), n = 6; Fig. 5] over bufferinjected controls. Injection of the inhibitor peptide at 4 h after insulin addition inhibited induction o f meiosis by about 40 min. Note that the cross-over from stimulation to inhibition is about 2 h (0.4 GVBD50) after insulin addition. These data suggest that PKC initially inhibits
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FIG. 6. Staurosporine inhibits Xenopus PKC and stimulates meiosis at concentrations above 0.1/aM. (A) Various concentrations of staurosporine were added to partially pure PKC [2] and the control samples received equivalent amounts of DMSO. After a 20rain incubation, PKC activity was measured (see Materials and Methods). Basal activity represents five determinations, whereas treatment groups are the average and S.E. of three values. Stars or asterisks represent a significant difference (= < 0.05). (B) Similar concentrations of staurosporine reduce PKC-dependent intracellular pH. Oocytes were incubated with various concentrations of staurosporine for I h then l U/ml PC-PLC and 14Cdimethyloxazolidine dione were added. After a second hour for radioisotope equilibration across the plasma membrane, the intracellular pH was determined. Each point represents at least four groups of
insulin induction of meiosis but, after about 0.4 GVBD50, is synergistic. We then examined the effect of another PKC inhibitor, staurosporine, on the ability of progesterone or insulin to induce meiotic cell division. First, we found that staurosporine inhibits Xenopus PKC activity after the enzyme was partially purified from oocytes by ionexchange resin [2]. The PKC activity eluted off in a sharp peak of activity at an ionic strength reported by Laurent et al. [2], was activated by phorbol ester, and peak activity fractions had greater amounts of a protein at 80,000 mol. wt. Staurosporine concentrations greater than 100nM were needed to inhibit Xenopus PKC (Fig. 6A). We examined whether staurosporine reduced intracellular pH as a measure of the drug's effect o n / n vivo PKC. After preincubation in various concentrations of staurosporine, 1 U/ml PC-PLC was added to the oocytes and intracellular pH determined. Staurosporine concentrations between 0.1 and 0.5/zM were required to reduce stimulated levels of intracellular pH (Fig. 6B) (possibly due to membrane damage or short life, inhibitor peptide did not alter the membrane distribution of the weak acid and our estimate of pH). Although purified PKC is more susceptible, there are six reports that micromolar levels of staurosporine are required for PKC inhibition in cellular extracts or to prevent cell proliferation (see [19], and Ref. [20] for a review). Similar concentrations of staurosporine stimulate progesterone-induced meiosis (Fig. 6C). Based on these dose-response data, 1 #M staurosporine was chosen as a minimal concentration that stimulates meiosis but would be less likely to inhibit enzymes other than PKC. Addition of staurosporine with insulin or up to about 3h after insulin (0.4-0.5 GVBD50) stimulates insulin (Fig. 7A). However, when the
five oocytes. (C) Progesterone (5 #M) induction of meiosis is accelerated by addition of 1/aM staurosporine (data represent three experiments). Control values (closed triangle), 0.1/aM staurosporine (open circle), and !/aM staurosporine (closed circle) are shown.
Protein kinase C and meiosis
(A)
potentiates hormone-induced DAG increases; thus, the stimulation of meiosis by staurosporine may reflect overproduction of DAG and stimulation of enzymes that depend on DAG. Various concentrations of the drug were added to groups of 10 ooeytes and DAG levels measured. After a 2 h incubation period, 0.1, 1 and 5 #M staurosporine did not increase DAG levels (two experiments; four determinations at each concentration). In addition, the drug had no effect on progesterone-induced early increase in DAG levels (data not shown).
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FIG. 7. Staurosporine data suggest that PKC is inhibitory early although insulin action may require PKC activity after about 0.5 GVBD50. (A) Early addition of 1/zM staurosporine stimulates but late addition inhibits insulin (2 #M) induction of meiosis. Results with control group (open circle), and groups with staurosporine addition at I h (closed circle), 2 h (open triangle), 3 h (closed triangle; 0.4 GVBD50), 4h (open square; 0.53 GVBD50), and 5h (closed square) after insulin addition are shown. (B) Addition of staurosporine always stimulates progesterone induction of meiosis. Control values (open circle), 1 #M staurosporine addition at 1 h (open triangle), 1.5 h (closed triangle) and 2 h (open square) after progesterone are shown. Insulin and progesterone data represent three experiments each.
drug was added after about 0.5 GVBD50, it inhibited insulin-induced meiosis. Staurosporine addition with progesterone stimulates progesterone-induced meiosis and maximal stimulation is achieved after about 0.3 GVBD50 (about 1.5h after progesterone addition; Fig. 7B). The use of inhibitors is plagued by the problem of non-specificity [21]. For example, Bishop et al. [22] have found that staurosporine
We conclude that PKC inhibits initiation of the processes leading to entry into prophase. Two PKC inhibitors stimulate and one activator inhibits (a total of six treatments with insulin and progesterone) the induction of meiosis when used before about 0.4 GVBD50. Note that this time point is 1.6-2.4h after hormone addition and that prophase begins at 4--6 h. These results agree with earlier reports that PKC inhibits meiotic cell division [6, 16]. We also suggest that PKC promotes the induction of meiosis after about 0.4 GVBD50. Other reports suggest this duality in that phorbol esters will initially enhance or inhibit a response and, when added at later times, produce the opposite effect [23-26]. Protein kinase C inhibition of cell division
PKC may be involved in checkpoints which review commitment to cell division [27]. As growth factors must be present for a period of hours before induction of cell division, this PKC inhibition may be responsible. The inhibitory mechanism may involve a decrease in G protein or phospholipase C activity and a reduction in agonist-stimulated phosphoinositide turnover [22, 28-30]. However, the breakdown of polyphosphoinositide 4,5bisphosphate (PIP2) and calcium release may not be required for growth factor-induced proliferation in many cells including oocytes [1, 11, 29-35].
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A second possible mechanism of PKC inhibition of meiosis could be related to phorbol ester stimulation of adenylate cyclase [28, 36]. PKC could increase cAMP to inhibit proliferation since, in the oocyte, high cAMP levels are inhibitory (see Ref. [37]). However, I/~M phorbol ester has little or no effect upon cAMP levels in Xenopus oocytes [38]. Phorbol ester inhibited meiosis in mouse oocytes yet was unable to prevent the initial decrease in cAMP [39]. These studies suggest that the PKC inhibition of meiotic induction occurs at a point distal to the decrease in cAMP-dependent protein kinase A activity.
Late PKC activity may be predominantly synergistic We also propose that PKC becomes synergistic to the induction of meiotic cell division after about 0.4 GVBD50. At this time (a) the presumptive PKC activator (PC-PLC) stimulates progesterone-induced meiosis, (b) PC-PLC is less inhibitory to insulin-induced meiosis, (c) staurosporine inhibits insulin-induced meiosis, (d) inhibitor peptide injection inhibits insulin action. PKC may be involved in late events that take place from about 0.4 GVBD50 until white spot appearance since DAG again increases over this period and peaks at 0.85 GVBD50 for insulin, progesterone, or insulin-like growth factor 1 [7] (see also Introduction). The change in PKC action may be about 0.4 GVBD50 since at this point (a) staurosporine's effect on insulin switches from stimulation to inhibition, (b) staurosporine reaches maximal stimulation of progesterone, (c) PC-PLC's effect on progesterone switches from inhibition to slight stimulation, and (d) inhibitor peptide stimulation switches to inhibition of insulin action at this point. Thus, three late treatments and the trend shown by inhibitor peptide on progesterone action support the idea of late PKC synergism. However, two late treatments do not: (1) late addition of staurosporine does not inhibit progesterone action, and (2) late addition of PC-PLC does not stimulate insulin action.
These findings could be due to an effect of PCPLC or staurosporine on a pathway not involving PKC. The ability of PC-PLC to stimulate late progesterone but not insulin action may be related to the ability of PKC to phosphorylate and inhibit the insulin and IGF1 receptors [40--43]. At this time, there are no demonstrated effects of PKC on the progesterone receptor in oocytes.
Use of PKC inhibitors and activators Inhibitor peptide is relatively specific to PKC as 2115-fold higher amounts are required to inhibit cAMP-dependent protein kinase A [18]. As opposed to sphingosine [6, 16], this PKC inhibitor alone did not induce meiosis. Staurosporine was also used due to its ability to inhibit PKC [44] and it produced results similar to the inhibitor peptide. As evidence that the drug acts through PKC inhibition, staurosporine concentrations required to decrease intracellular pH were similar to those that were required to effect induction of meiosis. Surprisingly, this PKC inhibitor has been found to be a tumour promoter [45]. Based on the work presented here, this turnout promotion may be due to its ability to reverse the PKC inhibition of the initiation of cell division. In an attempt to avoid the use of phorbol esters and to provide data that can be compared to that obtained by inhibitors, we used PC-PLC to increase DAG and stimulate PKC. Due to the inaccessibility of some phospholipids such as PIP2 to externally added phospholipase [46] and the specific nature of PC-PLC, addition of highly pure PC-PLC to oocytes should only produce effects through increased DAG. In agreement with this belief is our finding that PC-PLC did not cause an increase in intracellular calcium that would represent breakdown of PIP2 to IP3 or general membrane disruption. Finally, PC-PLC may be acting through DAG and PKC as similar concentrations of the enzyme increase DAG, intracellular pH (a PKC-del~ndent process) and affect the induction of meiotic cell division. Further evidence that PC-PLC acts through
Protein kinase C and meiosis PKC activation is found i n the literature as addition of 0.5 U/ml phospholipase C mimics phorbol ester action on rat follicle-enclosed oocytes [47] or hepatocytes [48]. Apparent contradiction: P K C induction and inhibition o f meiosis
Treatments that increase D A G levels (ras p21 microinjection [10]; D A G kinase inhibitor [49]; insulin, IGF-1 or progesterone addition [7]) or addition of a phorbol ester [1] induce meiosis. However, this report demonstrates an initial PKC inhibition of meiosis. This apparent contradiction of PKC action is reflected in the literature. Bornslaeger et al. [39] have shown that phorbol esters inhibited meiosis in mouse oocytes, but Aberdam and Dekel [47] found that phorbol esters stimulated meiosis in follicle-enclosed rat oocytes (see Ref. [50] for a review). Phorbol esters induce meiosis in Spisula yet inhibit 5-hydroxtryptamine-induced meiosis in this cell [51]. In at least three other cell systems, phorbol esters induce proliferation alone yet antagonize growth factor action in the same cell [52]. Phorbol esters induce proliferation in fibroblasts and epithelial cells, act as comitogens in lymphocytes, yet inhibit mitogenesis in muscle and some epithelial cells (see Ref. [53] for a review). Phorbol esters inhibit proliferation of undifferentiated carcinoma cells and stimulate proliferation of differentiated derivatives of these cells [54-56]. Differences in phorbol ester action could be due to the ability of the phorbol esters to stimulate and then down-regulate PKC (i.e. phorbol esters may induce meiosis by actually depleting the oocyte of PKC). This may explain the inability of injected PKC to induce meiosis [1, 4]. One may also suggest that the apparent discrepancy between PKC induction and inhibition is related to the early PKC inhibition and a late PKC stimulation of cell division reported here. Phorbol esters may selectively induce the PKC synergistic pathway whereas PC-PLC and staurosporine affect the inhibitory pathway. As Stith et al. [7] have suggested that the two hormone-induced D A G peaks occur in
401
different locations within the cell, inhibition and stimulation may be due to PKC action in different locations. Different isozymes of PKC may be responsible for the inhibition or stimulation since there are two isozymes of PKC in Xenopus oocytes [57]. Protein kinase C in the induction o f meiotic cell division
Based on the above discussion, a refinement of the model for PKC involvement in the induction of meiotic cell division can be proposed. Insulin acts through the IGF-1 receptor [58] and through ras p21 [59, 60] to increase D A G levels [7, 11]. Progesterone also induces an increase in D A G levels. The resulting activation of PKC induces various proliferative events that may not be required for cell division (such as the increase in intracellular pH [1]) but the overall effect of PKC at this time is inhibition. The period of PKC inhibition is relieved at about 2 h after hormone addition and synergistic PKC pathways predominate. Independent PKC inhibitory and stimulatory paths may be due to the presence of an active enzyme in different subcellular locations or to different PKC isozymes. Acknowledgements--Thanks to CHRISJAYNESand SALLYSILVAfor assistance. This work was supported in part by NSF grant DCB-8916703.
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