BIOLOGY OF REPRODUCTION 48, 846-850 (1993)
Involvement of Protein Kinase C in Agonist-Stimulated Goldfish Ovulation'
FREDERICK WILLIAM GOETZ 2
University of Notre Dame, Department of Biological Sciences, Notre Dame, Indiana 46556
ABSTRACT
The effects of two protein kinase C (PKC) inhibitors, calphostin C and staurosporine, on the in vitro ovulation of goldfish
(Carassius auratus) oocytes were investigated. Ovulation was stimulated by prostaglandin (PG) F2, (PGF,,, 2.0 ,ug/ml), by
sodium orthovanadate (0.1 mM), by a combination of the phorbol ester phorbol 12-myristate-13-acetate (PMA, 0.1 utg/ml) and
calcium ionophore A23187 (0.05 Itg/ml), by thapsigargin (0.2 jig/ml), and by elevated pH (8.1). In addition, the effects of
these inhibitors on the PKC activity of the goldfish follicle wall was determined by use of a specific peptide substrate phosphorylation assay. At 0.1 JiM, staurosporine significantly blocked ovulation induced by all agents. However, at lower (0.01 jtM)
levels it blocked only PMA/A23187-induced ovulation. In contrast, calphostin significantly blocked only PMA/A23187-induced
ovulation, although there was a decrease in pH-induced ovulation at lower calphostin concentrations. Both calphostin and staurosporine blocked follicular PKC activity at levels that were inhibitory to ovulation In addition, staurosporine significantly blocked
PKC activity at levels even lower than those needed to block ovulation. The combined results suggest that orthovanadate, PGF,.,
and thapsigargin do not require PKC activation for the induction of ovulation, whereas PMA/A23187 does.
INTRODUCTION
Several mediators have now been reported to stimulate
in vitro ovulation of goldfish (Carassiusauratus) oocytes.
Prostaglandins were the first agents reported to induce ovulation of goldfish follicles in vitro [1]. Following this, it was
demonstrated that the pH of the medium used to incubate
goldfish follicles was also capable of stimulating ovulation
[2]. Thus, in the absence of any agonists, there was a progressive increase in the amount of ovulation as the pH of
the medium became more alkaline. It appeared that this
effect was mediated through the stimulation of prostanoid
synthesis since indomethacin strongly inhibited it.
More recently, several studies were directed at determining the signal transduction pathways involved in the
control of goldfish ovulation. In the course of these studies,
it was shown that the phorbol ester phorbol-12-myristate13-acetate (PMA) and the calcium ionophore A23187 synergistically stimulate goldfish ovulation in vitro [3]. Synthetic diacylglycerols, such as diC 6 and diC8, also synergize
with A23187 in stimulating ovulation. Since these agents are
known to stimulate protein kinase C (PKC), these results
suggested that ovulation could be stimulated through the
activation of PKC. It has been recently reported that the
general G protein activators, sodium orthovanadate and
fluoroaluminate, stimulate goldfish ovulation and phosphatidylinositol (PI) cycling in the follicle wall [4]. These effects
appear to be general characteristics of a number of oxoanionic compounds [5]. Finally, in this paper we report that
in vitro ovulation can also be stimulated with thapsigargin,
an agent that increases cytosolic Ca2 +, presumably by specifically inhibiting the sequestration of Ca 2+ by cellular organelles [6].
Given the previous results with orthovanadate, and since
the natural stimulators of PKC are second messengers arising from PI cycling, we hypothesized that orthovanadate
stimulates ovulation through PI cycling, followed by the activation of PKC. If this is true, then we should be able to
block the stimulatory effect of orthovanadate on ovulation
by using PKC inhibitors. In the present study we report the
effects of several PKC inhibitors on orthovanadate- and PMA/
A23187-stimulated ovulation, and we attempt to relate these
results to the actual inhibition of follicular PKC. In addition,
we also studied the effects of these PKC inhibitors on the
stimulation of ovulation by prostaglandin (PG) F2,, (PGF2a),
thapsigargin, and alkaline pH since the mechanism by which
these agents stimulate ovulation is unknown.
MATERIALS AND METHODS
Experimental Animals and In Vitro Ovulation Assays
Gravid goldfish (30-50 g) were purchased from Ozark
Fisheries (Stoutland, MO) prior to the reproductive season
and maintained under long photoperiods (16L:8D) in circular flow-through tanks supplied with 12.5 0C well water.
Experiments were conducted from mid-April to early July.
Sexually mature females were chosen on the basis of the
size of the oocytes, sampled in vivo as previously described
[2]. Fish with oocytes greater than 1 mm in diameter were
induced to undergo germinal vesicle breakdown (GVBD)
by a combination of warm temperature (20°C) and the injection of hCG (5 or 10 IU/g BW). Generally, GVBD occurs
in vivo 5-7 h after hCG injection, and in the present study
the ovary was removed for in vitro incubations approximately 1-2 h after GVBD. Since the follicles were obtained
well after oocyte maturation, the effects of the agonists that
were investigated would presumably be on follicular rupture and/or oocyte expulsion. Fish were killed after being
anesthetized with 2-phenoxyethanol. The ovary was re-
Accepted December 16, 1992.
Received September 10, 1992.
'This work was supported by NIH grant #HD25924-02.
2
Correspondence. FAX: (219) 239-7413.
846
847
PROTEIN KINASE C AND GOLDFISH OVULATION
moved and individual follicles were detached from extrafollicular tissue by gently pipetting the ovarian tissue in
goldfish Ringer's solution [7] containing Tris (0.025 M) at
pH 7.3. Mature follicles were separated from immature oocytes and connective tissue under a dissecting microscope.
For in vitro incubations, replicates of 10 mature follicles
were then transferred to wells of Falcon or Corning polystyrene tissue culture plates (24-well) containing 1.0 ml of
medium with or without test agents, and were incubated at
20°C under standard fluorescent room lights (800 lux). Within
the experiments conducted on each fish, three replicates
of 10 follicles each were tested per treatment. At the time
of assay, ovulation was determined in all incubates by
counting the spent follicle walls and oocytes with swollen
chorions.
Agents used to stimulate ovulation included PGF 2a (2.0
,ug/ml; Sigma Chemical Co., St. Louis, MO), orthovanadate
(0.1 ,aM; Sigma), thapsigargin (0.2 ,ag/ml; LC Services,
Woburn, MA), and a combination of PMA (0.1 pg/ml; Sigma)
and A23187 (0.05 jig/ml; Calbiochem, LaJolla, CA). Ovulation was also stimulated by an elevated pH of 8.1, obtained
by adjusting the ratio of Tris base and acid in the medium.
Concentrations of orthovanadate, PMA/A23187, and PGF2,
were based on past studies and represent levels that just
stimulate maximal ovulation [2-4]. Since the effects of thapsigargin on ovulation have not been reported, a dose-response test was run and the results are presented here. The
PKC inhibitors tested were staurosporine (0.1 and 0.01 pM;
Kamiya Biomedical, Thousand Oaks, CA) and calphostin C
(1.0 and 0.1 pIM; Kamiya and LC Services). Concentrations
of these inhibitors were based on preliminary experiments
and also on reported inhibitory constants for these agents.
Stocks of PGF2, and thapsigargin were prepared at high
concentrations in 95% ethanol, and an aliquot was dissolved in the medium to obtain the desired final concentration. All other agents were initially dissolved in high concentrations in dimethyl sulfoxide (DMSO), and appropriate
aliquots were added to the incubation medium to obtain
the desired final concentration. Given the combination of
treatments, some incubates treated with both a stimulator
and an inhibitor may have had a maximum of 0.2% DMSO.
This would have been the case for PMA/A23187 incubations containing one of the PKC inhibitors. All other incubations would have had less vehicle or a combination of
0.1% ethanol and 0.1% DMSO.
PKC Assay
PKC was assayed with use of a kit (Amersham Corp., Arlington Heights, IL) that measured the transfer of the gamma
phosphate of 32 P-labeled ATP to a peptide substrate specific
for PKC. The sequence of the peptide, NH 2-Arg-Lys-Arg-ThrLeu-Arg-Arg-Leu-COOH, was based on the PKC phosphorylation site of the epidermal growth factor receptor [8,9].
Tissue samples were prepared by transferring 100 follicles to 1.0 ml of ice-cold medium (as above) in a dispos-
able micro-glass tissue homogenizer. The yolk was then
gently squeezed from the follicle by pressing the homogenizer to the bottom of the tube several times. The solution
containing the tissue was transferred to a 1.5-ml microcentrifuge tube and spun at 3000 rpm at 40C. The supernatant
was removed, and the pellet was washed three times, each
time with 1.0 ml of ice-cold medium. The solution was centrifuged at 3000 rpm after each wash. After removing the
supernatant following the final wash, the pellet was homogenized with a micropestle in 100 Il of a sample buffer
containing 50 mM Tris/HCl at pH 7.5, 5.0 mM EDTA, 10.0
mM EGTA, 0.3% mercaptoethanol, 10 mM benzamidine, and
50 pIg/ml phenylmethylsulphonyl fluoride. The homogenate was centrifuged at 3000 rpm in the cold for 10 min,
and the supernatant was removed. In the case of experiments on the effects of PKC inhibitors, the inhibitor was
included in the sample buffer prior to homogenization.
PKC assays were carried out by mixing, in a 1.5-ml microcentrifuge tube, 25 l1 of a 50-mM Tris/HCl (pH 7.5)
solution containing 3.0 mM calcium acetate, 2 mole % of
L-ot-phosphatidyl-L-serine, 6 pzg/ml phorbol-12-myristate-13acetate, 7.5 mM dithiothreitol, 225 p.M of specific peptide
substrate (as described above), and 0.05% sodium azide;
25 p.l of tissue sample (as described above); and 25 1 l of
a 50 mM Tris/HCl (pH 7.5) solution containing 150 ,aM
ATP, 45 mM magnesium acetate, 0.05% sodium azide, and
approximately 0.75 Ci y-[ 3 2P]-labeled adenosine 5'-trisphosphate, tetra-triethylammonium salt (4000 Ci/mmol; ICN
Biomedicals, Costa Mesa, CA). The assay was incubated at
25°C for 25 min directly under a lamp holding two 15-watt
Philips Cool White (F15T8/CW) fluorescent bulbs (2500 lux).
After incubation, the reaction was terminated by adding 100
Rl1of a dilute ortho-phosphoric acid solution. After mixing,
125-pl aliquots were placed on 2.5-cm 2 phosphocellulose
papers to separate the peptide substrate from free ATP. The
papers were then washed twice in 10% glacial acetic acid
for 10 min/wash. After the wash, the papers were placed
in 20-ml scintillation vials, and 10 ml of Formula 989 scintillation fluid (du Pont deNemours and Co., Wilmington,
DE) was added. Samples were counted, and the amount of
phosphate transferred to the specific peptide/min was calculated for each sample after the specific activity of the
magnesium ATP solution was determined.
Data Analysis
Percent ovulation data was transformed by the arc-sin
transformation before statistical analysis. Transformed ovulation data was analyzed by one-way ANOVA followed by
Tukey's studentized range test. Data from the PKC enzyme
assay were analyzed by Student's t-test.
RESULTS
Effects of PKC Inhibitors on Agonist-lnduced Ovulation
Figure 1 shows the results for the effects of staurosporine and calphostin C on ovulation induced by orthovana-
848
GOETZ
FIG. 1. Effects of calphostin (CALPHOS) and staurosporine (STAUROS) on sodium orthovanadate- (ORTHOVAN) and PMA/A23187-induced
goldfish ovulation after 2 h of incubation. Each bar represents the mean
SEM for incubations conducted on the follicles of three fish, performed in
triplicate/fish. * Significantly different from corresponding treatment in the
absence of PKC inhibitor.
date and PMA/A23187. Only PMA/A23187-induced ovulation was blocked by calphostin, and this was significantly
different from controls at 1.0 IzM. In contrast, staurosporine
significantly blocked ovulation at 0.1 F.M in orthovanadatestimulated incubates and at 0.1 and 0.01-p.M concentrations
in the incubates containing PMA/A23187. Neither inhibitor
had a significant effect by itself.
At 2.0 and 0.2 pzg/ml, thapsigargin was a potent inducer
of in vitro ovulation, stimulating 83 ± 10% and 82 + 10%
ovulation, respectively (vs. controls; 18 + 11%; n = 4). At
0.02 p.g/ml the amount of ovulation induced by thapsigargin decreased to 46 ± 18%. Calphostin did not block the
effects of thapsigargin or PGF 2., whereas staurosporine at
0.1 p.M significantly inhibited ovulation induced by these
agents (Fig. 2). For incubations at pH 8.1, results at both 2
FIG. 3. Effects of calphostin (CALPHOS) and staurosporine (STAUROS) on ovulation induced by pH 8.1 medium after 2 h and 4 h of incuSEM for incubations conducted on
bation. Each bar represents the mean
the follicles of three fish, performed in triplicate/fish. * Significantly different from corresponding treatment in the absence of PKC inhibitor.
and 4 h are shown (Fig. 3), since ovulation induced by elevated pH generally requires a longer time than for other
agents. At 0.1 pIM, staurosporine significantly blocked pHinduced ovulation; however, the effect of calphostin was
more complex. While 1.0 LM calphostin had no effect on
pH-induced ovulation, the lower level (0.1 iM) appeared
to be inhibitory. Although this inhibitory effect was not statistically significant, it was consistent across the three fish
that were tested.
Effects of PKC Inhibitors on FollicularPKC Activity
The PKC activity in the goldfish follicle wall is illustrated
in Figure 4. There was detectable phosphorylation of endogenous proteins in the absence of the peptide substrate
(END); however, there was a significant increase in the PKC
activity in the presence of the substrate (CON). At concen.q
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FIG. 2. Effects of calphostin (CALPHOS) and staurosporine (STAUROS) on PGF2,- and thapsigargin- (THAPSIG) induced goldfish ovulation
after 2 h of incubation. Each bar represents the mean ± SEM for incubations conducted on the follicles of three fish, performed in triplicate/fish.
* Significantly different from corresponding treatment in the absence of
PKC inhibitor.
0.1 uM
0.1 uM .01 uM
.001 uM
FIG. 4. Follicular PKC activity in the absence of PKC inhibitors (CON)
and with calphostin (CAL) or staurosporine (STA). Phosphorylation of endogenous protein in the absence of tho specific peptide substrate is indicated by bar labeled END. Each bar represents the mean - SEM for incubations conducted on the follicles of three fish, performed in duplicate/
fish. * Significantly different from control in the absence of inhibitor.
PROTEIN KINASE C AND GOLDFISH OVULATION
trations that blocked ovulation, calphostin (1.0 ,uM) and
staurosporine (0.1 and 0.01 IpM) significantly decreased this
PKC activity. In addition, in the case of staurosporine, but
not calphostin, even lower concentrations appeared to inhibit PKC activity.
DISCUSSION
The results of the present study indicate that there is a
differential effect of calphostin C and staurosporine on ovulation induced in goldfish follicles by various agonists or
incubation conditions. Calphostin and staurosporine are two
of a growing number of purported PKC inhibitors that have
become popular in the literature. While staurosporine is
certainly a very potent PKC inhibitor, it is not very specific
(see, e.g., [10]). Thus, it can block other kinases, complicating the interpretation of its effects. In contrast, calphostin
C is a more specific inhibitor of PKC [11], but it now appears that its potency is dependent on light activation [12],
and this activation has not been well characterized. Thus,
the physical parameters surrounding its use could drastically alter the final results obtained. In the experiments described here, all follicle incubations were conducted under
standard fluorescent room lighting, and the PKC assays were
carried out under direct fluorescent light of higher intensity. In preliminary experiments with the PKC assay, we did
observe that calphostin inhibited PKC activity only weakly
without this lighting (results not shown).
Past studies have shown that goldfish ovulation can be
induced by various PKC stimulators such as PMA, and by
synthetic diacylglycerols [3]. Further, several general G protein activators including orthovanadate and fluoroaluminate
can stimulate ovulation and inositol phosphate accumulation at similar agonist concentrations [4]. Thus, it seemed
reasonable to hypothesize that orthovanadate and fluoroaluminate induce ovulation by activating PI cycling, eventually resulting in PKC stimulation. However, the data presented here do not support this hypothesis. First, while
calphostin C, which is presumably a more specific PKC inhibitor, blocked PMA/A23187-stimulated ovulation, it was
unable to block orthovanadate-induced ovulation. Given that
vanadate would theoretically work upstream of PMA/A23187
(if our hypothesis was correct), it appears that if PMA/A23187
was blocked under these conditions then vanadate-induced
ovulation should also be. Second, staurosporine at both 0.1
and 0.01 pzM was able to significantly block follicular PKC
activity to an extent that exceeded calphostin inhibition, regardless of the calphostin level. However, the lower level
of staurosporine (0.01 ,uM) was unable to block orthovanadate-induced ovulation although it significantly blocked
ovulation induced by PMA/A23187. Thus, it appears that orthovanadate stimulates ovulation by a PKC-independent
mechanism.
Vanadate is believed to affect the activities of various
phosphoryl transfer enzymes, presumably by adopting a
849
structure resembling the transition state for phosphoryl
transfer reactions [13]. Thus, it could affect a number of
processes requiring phosphorylation. Vanadate has been
shown to be a potent inhibitor of Ca2+-ATPase and the
transport of calcium across cellular membranes [14], and
this could be an indirect means by which it stimulates ovulation. Inhibiting the calcium pump would allow calcium
to move across the plasma membrane or out of internal
organelles and to increase in the cytoplasm. This increased
Ca 2+ could then stimulate the contraction of smooth muscle-like cells in the follicle, resulting in oocyte expulsion
(see below). As with orthovanadate, calphostin was also unable to block ovulation induced by PGF2,, or thapsigargin,
implying that neither of these requires PKC activity for the
induction of ovulation. However, preliminary experiments
we have conducted, in which follicles have been preincubated with higher levels of PMA alone in an attempt to downregulate PKC activity prior to agonist stimulation, indicate
that PKC may be involved in PGF-induced ovulation. In contrast, down-regulation of PKC with PMA has no effect on
orthovanadate or thapsigargin-induced ovulation, supporting the inhibitor data presented here. Thus, the result with
PGF2a must be cautiously interpreted.
Although calphostin did appear to block pH-induced
ovulation, the results are not easy to interpret since, in contrast to PMA/A23187 incubates, only the lower level of calphostin was inhibitory. The induction of ovulation by pH
is very strongly inhibited by indomethacin [2], indicating
the involvement of some cyclo-oxygenase product. Thus,
we expected to see results similar to those obtained with
PGF2 ,. However, if pH induces the synthesis of some eicosanoid, then the effect of the protein kinase inhibitor may
be more complex, since it could influence not only the action, but also the synthesis, of the eicosanoid. Interestingly,
staurosporine has been shown to have dichotomous effects
at different concentrations that may be related to its effects
on eicosanoid synthesis. In macrophages, staurosporine
stimulated PGE 2 formation and arachidonic acid release at
low concentrations, while higher levels did not [15]. However, we know of no similar reports for calphostin.
The fact that higher levels (0.1 ,uM) of staurosporine
blocked ovulation induced by all agonists suggests that at
these levels it might inhibit other essential kinases involved
more generally in ovulation. A good candidate would be
myosin light-chain kinase (MLCK). Staurosporine has been
reported to be a very potent inhibitor of MLCK [16]. MLCK
is known to phosphorylate myosin light chains in vertebrate smooth muscle, and this is the signal for activation of
cross-bridging and thus contraction [17]. Inhibition of this
kinase would certainly block ovulation if smooth musclelike contraction is necessary for oocyte expulsion. Interestingly, MLCK is activated by Ca2+ , and this could be another
way that vanadate influences oocyte expulsion. Staurosporine has been reported to block ovulation in rats when applied intrabursally before or after hCG induction [18]. How-
850
GOETZ
ever, at the effective concentration (10 tLM), staurosporine
would undoubtedly inhibit a variety of kinases including
MLCK.
Finally, this is the first report of the effects of thapsigargin on ovulation. Thapsigargin is a plant-derived sesquiterpene lactone that reportedly specifically inhibits the uptake
of Ca2+ into intracellular storage areas [6]. The mechanism
for this inhibition appears to be the selective blockage of
intracellular Ca2+-ATPases [191. In the present study, thapsigargin was a very potent stimulator of ovulation. In contrast, the calcium ionophore, A23187, does not appear to
be very potent by itself in stimulating ovulation, though it
is potent in the presence of PMA [3]. This is curious since
the ultimate effect of both of these would seem to be the
elevation of intracellular Ca2 +, regardless of their specific
mechanism of action. Another interesting observation concerning the stimulation of ovulation by thapsigargin is that
it is ineffective in the absence of external Ca2+ (results not
shown). Thus, the results suggest that follicular rupture induced by thapsigargin requires Ca 2+ from both intracellular
and extracellular sources.
In summary, this study confirms that PMA/A23187-stimulated ovulation occurs by the stimulation of PKC. In addition, the results strongly suggest that ovulation induced
by orthovanadate or thapsigargin do not require PKC activation.
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