From the
The role of sphingomyelin-derived second messengers in
progesterone-induced reinitiation of the meiotic cell cycle of
Xenopus laevis oocytes was investigated. A brief treatment of
defolliculated oocytes with sphingomyelinase (Staphylococcus
aureus) was sufficient to induce maturation as measured by H1
kinase activity and germinal vesicle breakdown (GVBD). Pretreatment
with cycloheximide inhibited sphingomyelinase-induced GVBD
demonstrating a requirement for protein synthesis. Microinjection of
ceramide or sphingosine, potential products of sphingomyelin
hydrolysis, were capable of inducing GVBD in the absence of hormone.
Metabolic labeling studies suggested the conversion of sphingosine to
ceramide was necessary for sphingosine-induced GVBD. Additionally,
fumonisin b
While the induction of oocyte maturation by progesterone has
been widely studied, the mechanism of reinitiation of the meiotic cell
cycle is poorly understood (Matten and Vande Woude, 1994). Unlike the
classical steroid hormone pathway for transcriptional activation, there
is evidence, including the recent identification of a putative high
affinity receptor (Liu and Patino, 1993), that progesterone, released
from surrounding follicle cells, initiates its biological effect at the
plasma membrane of amphibian oocytes (Godeau et al., 1978;
Smith and Ecker, 1971). This has led to numerous studies implicating
lipid-derived second messengers in the transmembrane signaling
mechanism of progesterone (Stith and Maller, 1987; Chien et
al., 1991; Kostellow et al., 1993; Carnero and Lacal,
1993). While progesterone has been observed to stimulate the turnover
of the major glycerophospholipids of oocytes with the generation of
potential lipid second messengers, these are not sufficient to induce
maturation in the absence of hormone. In contrast, the generation of
sphingomyelin-derived second messengers may be crucial components of
the maturation pathway (Varnold and Smith, 1990a, 1990b). Varnold and
Smith have previously reported that treatment of oocytes with bacterial
sphingomyelinase was a potent inducer of maturation. They additionally
showed that microinjection of sphingosine was sufficient to reinitiate
the meiotic cell cycle (Varnold and Smith, 1990a, 1990b). These
observations are consistent with the emerging role of
sphingolipid-derived second messengers in regulating critical cellular
processes (Hannun and Bell, 1989, 1993; Hannun, 1994). In particular,
the transient generation of ceramide from sphingomyelin, stimulated by
a variety of agonists, has recently led to the proposal of a new
signaling system used during monocytic differentiation of HL-60 cells
(Okazaki et al., 1989; Kim et al., 1991; Strum et
al., 1994) and programmed cell death (Obeid et al., 1993;
Jarvis et al., 1994). Therefore, we have further investigated
the role of sphingolipid-derived second messengers in
progesterone-induced maturation of Xenopus oocytes. In this
study, we identify the existence of a sphingomyelin cycle in
Xenopus oocytes, which is activated by progesterone.
Additionally, our results suggest that ceramide may be functionally
important in mediating the reinitiation of the meiotic cell cycle
triggered by progesterone.
There have been numerous reports demonstrating the generation
of lipid second messengers during progesterone-induced maturation of
Xenopus and Rana oocytes (Stith and Maller, 1987;
Varnold and Smith, 1990a, 1990b; Chien et al., 1991; Kostellow
et al., 1993; Carnero and Lacal, 1993). However, most studies
have concluded that while these second messengers may play a role in
the maturation pathway, they are not sufficient to induce maturation in
the absence of hormone. In contrast, Carnero and Lacal (1993) have
reported studies conducted to evaluate the role of DAG, phosphatidic
acid, lysophospholipids, and arachidonic acid in oocyte maturation.
These investigators microinjected bacterial phospholipase C, D, or
A
There are
several potential metabolites generated during sphingomyelinase
treatment of oocytes, which could be involved in inducing the
maturation pathway. Varnold and Smith (1990a, 1990b) proposed that
sphingosine, generated indirectly from sphingomyelin, was the
biologically active metabolite exerting its effect through the
inhibition of protein kinase C. This was partly based on the
observation that microinjected sphingosine induced GVBD. We also found
that microinjected sphingosine as well as phytosphingosine could induce
GVBD. However, our observations that sphingosine is rapidly metabolized
to ceramide upon microinjection into cells and that microinjected
ceramide can induce GVBD suggest that ceramide may be the more proximal
mediator of sphingomyelinase-induced maturation. Futhermore, in support
of ceramide as the mediator, the sphingosine N-acyltransferase
inhibitor fumonisin B
A variety of agonists
including the steroids 1,25-dihydroxyvitamin D
, an inhibitor of sphingosine
N-acyltransferase, blocked sphingosine-induced GVBD
demonstrating that ceramide is the more proximal biologically active
metabolite. Treatment of oocytes with progesterone, the physiological
inducer of oocyte maturation, resulted in a time- and
concentration-dependent increase in the mass of ceramide and decrease
in the mass of sphingomyelin through activation of a
Mg
-dependent neutral sphingomyelinase. These
observations suggest that the generation of ceramide from sphingomyelin
is part of the signal transduction pathway activated in response to
progesterone and that the increase in ceramide is likely to be
functionally important in resumption of the meiotic cell cycle.
Materials
Progesterone, 10 mg/ml stock in
ethanol, sphingomyelinase (Staphylococcus aureus), tricane
(3-aminobenzoic acid ethyl ester methane sulfonate salt, bisbenzimide
H33342 fluorochrome, fumonisin B, pregnant mare serum
gonadotropin, and gentamycin were from Sigma. DAG
(
)
kinase was prepared as described previously (Loomis et
al., 1985).
[methyl-
C]Sphingomyelin (54.5
mCi/mmol),
[sphingosine-3-
H]N-hexanoylsphingosine
(10-30 Ci/mmol), and [
-
P]ATP (3000
Ci/mmol) was purchased from DuPont NEN. Xenopus laevis frogs
were from Nasco. Fatty acid-free BSA, phosphatidylcholine-specific
phospholipase C (Bacillus cereus), and phospholipase D
(cabbage) were from Boehringer Mannheim. Dioctanoylglycerol,
sphingosine, phytosphingosine, stearylamine, ceramides (bovine brain)
1,2-dioleoyl-sn-glycero-3-phosphocholine were from Avanti
Polar Lipids. 1,2-Dioleoyl-sn-glycerol was prepared by
phospholipase C hydrolysis of the corresponding phosphatidylcholine.
C
-ceramide, C
-ceramide,
erythro-N-acetyldihydrosphingosine,
threo-N-acetyldihydrosphingosine were prepared as described
previously (Van Veldhoven et al., 1989). All lipids were
>95% pure as judged by comigration with authentic standards on
thin-layer chromatography. Silica gel H plates were from Analtech.
Silica gel 60 plates were from Merck.
erythro-D-[4,5-
H]Dihydrosphingosine
(112 Ci/mmol) was the generous gift of Dr. Steve Wyrick at the
University of North Carolina at Chapel Hill. Poly(2-hydroxyethyl
methacylate) 12% in ethanol was obtained from Polysciences, Inc.
(Warrington, PA). All solvents were reagent grade or better.
Preparation of Oocytes
Female Xenopus frogs were primed by injection of pregnant mare serum gonadotropin
(100 units) into the dorsal lymph sac 3 days prior to isolation.
Ovaries were surgically removed from tricane-anesthetized frogs and
dissected into small tissue sections and placed in MB minus calcium
buffer (88 mM NaCl, 1.2 mM KCl, 2.4 mM
NaHCO, 15 mM HEPES, 0.8 mM
MgSO
, pH 7.6). Follicle cells were removed by incubating
oocytes for 1-2 h with collagenase (1 mg/ml) dissolved in MB plus
calcium (MB containing 0.32 mM CaNO
, 0.4
mM CaCl
). Following treatment, the cells were
extensively washed and then incubated in MB plus calcium. Stage VI
oocytes (1-1.2 mm in diameter) were selected for experimental
use. Samples of isolated oocytes were routinely stained with 1:1000 (1
mg/ml) bisbenzimide H33342 fluorochrome to visualize the extent of
removal of the follicle cells. Oocytes were visualized by fluorescent
microscopy and were found to be free of contamination by follicle
cells. Oocytes were stored on polyheme-coated dishes in MB plus
Ca
at 20 °C. Cells were used within 18 h of
isolation. Germinal vesicle breakdown (GVBD) was scored as the
formation of a small white spot in the animal hemisphere. Oocytes were
routinely fixed in 10% trichloroacetic acid and dissected to verify the
absence of the germinal vesicle.
Treatment of Oocytes with Exogenous
Phospholipases
Oocytes with an intact vitteline envelope were
treated with sphingomyelinase (S. aureus), phosphatidylcholine
phospholipase C (B. cereus), or phospholipase D (cabbage) at
the indicated concentrations for various periods of time.
Sphingomyelinase and phospholipase C were devoid of contaminating
activities as judged by in vitro assays. Also, the treatment
of oocytes with sphingomyelinase did not result in an increase in the
mass of diacylglycerol demonstrating the absence of a significant
contaminating phospholipase C activity. Cells were washed extensively
with MB containing 1 mM EDTA and 0.1% BSA. Cells were
resuspended in MB plus Ca.
H1 Kinase Assay
Oocytes were incubated with
progesterone (10 µg/ml final concentration) diluted from a 10 mg/ml
stock solution dissolved in ethanol and stored at -20 °C or
sphingomyelinase (S. aureus) at 0.25 units/ml for 5 min, at
which time the cells were extensively washed with MB containing 0.1%
BSA and 1 mM EDTA and then incubated at 18 °C in MB plus
calcium. At the time of GVBD and successive time points, oocyte samples
(5 cells each) were collected into ice-cold buffer containing 380
mM NaCl, 50 mM MgCl, 30 mM
Na
SO
, 10 mM KCl, 2 mM
NaHCO
, 20 mM HEPES, 1 mM EGTA, pH 7.4,
with NaOH; aspirated; and flash-frozen in liquid nitrogen. Oocytes were
thawed by homogenization (20 µl/oocyte) in oocyte lysis buffer (20
mM HEPES, pH 7.5, 80 mM
-glycerophosphate; 15
mM MgCl
, 20 mM EGTA, and 50 mM
NaF) to which Na
VO
(1 mM final
concentration) was added as well as a mixture of protease inhibitors
(15 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml each of
chymostatin, leupeptin, antipain, pepstatin A). Homogenates were
cleared by centrifugation (16,000
g) for 5 min at 4
°C, and the supernatants were recovered. For H1 kinase assays, 10
µl of the supernatant was added to 10 µl of kinase assay buffer
containing 40 mM HEPES, pH 7.3, 10 mM EGTA, 20
mM MgCl
, 0.2 mg/ml histone H1, 0.2 mM
ATP, 10 µM protein kinase A peptide inhibitor (peptide
TTYADFIASGRTGRRNAIHD), and 0.5 µCi/µl
[
-
P]ATP (3000 Ci/mmol) and incubated for 10
min at 23 °C. The reaction was stopped by the addition of 80 µl
of SDS sample buffer, and radiolabeled products were resolved by
SDS-polyacrylamide gel electrophoresis and detected by autoradiography.
Radiolabeled gel bands were excised and quantitated by liquid
scintillation counting.
Lipid Extraction and Quantitation
Groups of 20
oocytes treated with progesterone or vehicle were collected in liquid
nitrogen at various times. Total lipids were extracted as described
previously (Bligh and Dyer, 1959). Phosphatidylcholine and
sphingomyelin were resolved by thin-layer chromatography on Silica Gel
H plates in a solvent system consisting of chloroform/methanol/ammonium
hydroxide (65:35:8 (v/v/v)). Lipids were recovered from the silica gel
using a mixture of methanol/chloroform/HO (2.5:1.0:0.5
(v/v/v)) as described previously (Christiansen, 1975). Lipid phosphorus
was determined as described previously (Rouser, 1966). Oocytes
microinjected with [
H]hexanoylsphingosine or
[
H]dihydrosphingosine were extracted and resolved
on silica gel 60 plates in a solvent system of
chloroform/methanol/ammonium hydroxide (40:10:1 (v/v/v)). Lipids were
identified by comigration with known standards and quantitated by
liquid scintillation counting.
Quantitation of Diglycerides and Ceramides
The
total lipid extract was simultaneously assayed for diacylglycerol and
ceramide using DAG kinase as described previously (Preiss et
al., 1987). Briefly, one cell equivalent of total lipid extract
was passed over a 1-ml silicic acid column, washed with one column
volume of chloroform, dried under N, and resuspended in 20
µl of 3% Triton X-100. The reaction mixture, which consisted of DAG
kinase membranes (5 mg/ml) in a reaction buffer of 100 mM
imidazole, 100 mM NaCl, 25 mM MgCl
, 2
mM EGTA, pH 6.6, and 10 mM
[
-
P]ATP (100 cpm/pmol) in 100 mM
imidazole, pH 6.6, was added to a final volume of 100 µl. Samples
were incubated at room temperature for 30 min. The reaction was stopped
by the addition of 3 ml of methanol/chloroform (2:1 (v/v)) and 0.7 ml
of 1% HCl. [
P]Phosphatidic acid and
[
P]ceramide phosphate were obtained in the
chloroform phase following the addition of 1.0 ml of 1% HCl and 1.0 ml
of chloroform. The chloroform extract was washed with 2 ml of 1% HCl
twice to remove unreacted [
P]ATP. This extract
was dried under N
and resolved on silica gel 60 in a
solvent system of chloroform/acetone/methanol/acetic
acid:H
O (10:4:3:2:1 (v/v/v/v)).
[
P]Phosphatidic acid and
[
P]ceramide phosphate were localized by
autoradiography and comigration with authentic standards. Radiolabeled
lipids were quantitated by liquid scintillation counting.
Neutral Sphingomyelinase Assay
Neutral
sphingomyelinase activity of oocytes was assayed essentially as
described previously for HL-60 cells (Strum et al., 1994).
Oocytes were treated with progesterone for various periods of time and
collected in liquid N. Cells were resuspended in 100
mM Tris-HCl, pH 7.4, containing 0.1% Triton X-100 and 1
mM EDTA. The cell suspension was sonicated for 10 s using a
probe sonicator and centrifuged at 15,000
g for 5 min.
The supernatant was used as the enzyme source. The reaction mixture
contained cell lysate, 5 mM MgCl
, and 75
µM [
C]sphingomyelin. Samples were
incubated for 30 min at 37 °C in a shaking H
O bath.
Reactions were terminated by the addition of 3 ml of
methanol/chloroform (2:1 (v/v)) and 0.7 ml of H
O. Phases
were separated by the subsequent addition of 1.0 ml of chloroform and
1.0 ml of H
O. The aqueous phase was removed and counted by
liquid scintillation counting.
Microinjection of Oocytes
Lipids were dried in
Eppendorf tubes under N and resuspended in equal molar
amounts of fatty acid-free BSA in a buffer consisting of 20 mM
HEPES and 150 mM NaCl. Lipids were suspended at 1 mM,
and 40 pmol of lipids were injected into oocytes. Prepared in this
manner, this amount of lipid was found to be the minimum effective
concentration. Under these conditions, the time course of
sphingolipid-induced GVBD was variable among batches of oocytes. In
order to estimate the intracellular concentration of the lipid, the
volume of an oocyte was assumed to be 1 µl. Some oocytes were
microinjected with radiolabeled lipids complexed to BSA, collected in
liquid N
, and subsequently extracted and analyzed. Groups
of 20 oocytes were injected for each condition.
Treatment of Oocytes with Sphingomyelinase Induces
GVBD
Oocytes were treated exogenously with phospholipases to
determine if the generation of potential lipid second messengers could
stimulate GVBD or affect the timing of progesterone or insulin-induced
GVBD. Phospholipase C or D had little or no influence on the timing of
GVBD induced by insulin or progesterone and alone did not induce GVBD
(data not shown). However, as shown in Fig. 1A, a 5-min
treatment with S. aureus sphingomyelinase (0.25 units/ml) was
a potent inducer of GVBD with a time course similar to
progesterone-induced GVBD. The morphological appearance of
sphingomyelinase-treated oocytes undergoing GVBD was indistinguishable
from that of oocytes treated with progesterone, the physiological
inducer of maturation, suggesting that the normal processes of meiosis
were triggered. However, in response to some treatments, oocytes can
undergo GVBD as a degenerative process rather than a maturational
process (Smith, 1989). In order to confirm that the
sphingomyelinase-treated oocytes were undergoing normal meiotic
maturation, the levels of histone H1 kinase activity at successive
times following GVBD were assayed from sphingomyelinase-treated oocyte
cultures and compared with those obtained from oocytes treated with
progesterone. Histone H1 kinase activity, which is carried out by
activated p34 protein complexes, is required
for passage through the cell cycle and the levels of this activity
change over time during normal meiotic maturation in a characteristic
way. The levels of histone H1 kinase activity from both cultures were
similar at the time of GVBD and, at subsequent times, oscillated in a
similar manner (Fig. 1, B and C). These results
suggest that the sphingomyelinase-treated oocytes were triggered to
undergo the normal processes of meiotic maturation.
Figure 1:
p34 kinase activity
oscillates in sphingomyelinase-treated oocytes in a similar manner to
those treated with progesterone. Full-grown oocytes (40 each) were
either treated for 5 min with sphingomyelinase (, 0.25 units/ml)
followed by incubation in MB plus calcium or incubated in the
continuous presence of progesterone (
, 10 µg/ml dissolved
in MB plus calcium). The percentage of GVBD was determined for each set
of oocytes at successive time (panelA). Samples of
oocytes (5 oocytes each) were collected from each culture at the time
of GVBD and at specific times following GVBD and flash-frozen in liquid
nitrogen. Oocyte samples were thawed by homogenization in buffer and
assayed for p34 kinase activity. The products of the reaction
(
P-labeled histone H1) were resolved by SDS-polyacrylamide
gel electrophoresis and detected by autoradiography (panelC). The gel bands giving rise to the autoradiograph shown
in panelC were excised and counted, and the pmol of
[
P]phosphate incorporated was determined.
Results are shown in panelB for untreated oocytes
and *, sphingomyelinase
, progesterone
. The time
(in minutes) following GVBD at which the oocyte samples were collected
is indicated in panelC.
The effect of
varying concentrations of sphingomyelinase on the timing of GVBD was
examined. Fig. 2shows that the time course of GVBD was
significantly accelerated by incubation with increasing
sphingomyelinase concentrations. The GVBD of cells treated
with 0.5 units/ml was decreased by 2 h compared with cells treated with
0.1 units/ml. These data demonstrate that the timing of GVBD following
sphingomyelinase treatment is dependent on the concentration of
sphingomyelinase. Since sphingomyelinase hydrolysis of sphingomyelin
generates ceramide, a potential second messenger implicated in the
regulation of cell growth, differentiation, and apoptosis, we next
quantitated the mass of ceramide following treatment of oocytes with
sphingomyelinase, which was sufficient to induce maturation. As shown
in Fig. 3, treatment with 0.25 units/ml for 5 min caused a 25%
increase above basal levels. The ceramide increased steadily and at
GVBD
(3 h) represented a 75% increase over untreated
cells. This may indicate that some sphingomyelinase remained bound to
the oocytes following treatment or an endogenous sphingomyelinase was
activated. The mass of diacylglycerols did not increase upon
sphingomyelinase treatment of the cells, indicating that the
sphingomyelinase preparation did not contain a significant
contaminating phospholipase C activity. These data suggest that
relatively small changes (<2-fold) in the cellular content of
ceramide is sufficient to induce GVBD.
Figure 2:
Effect of sphingomyelinase concentration
on the time course of induction of GVBD. Groups of 20 oocytes were
treated with various concentrations of sphingomyelinase for 5 min.
Progesterone (), sphingomyelinase 0.1 units/ml (
), 0.3
units/ml (
), 0.5 units/ml (
), 0.6 units/ml (
), 0.8
units/ml (
), 1.0 unit/ml (+). Results are expressed as the
percentage of total cells which have undergone GVBD as measured by the
appearance of a white spot on the animal
pole.
Figure 3:
Changes
in ceramide mass in oocytes treated with sphingomyelinase. Oocytes were
treated with sphingomyelinase, 0.25 units/ml, for 5 min, washed, and
incubated in MB plus calcium. Cells were collected into liquid nitrogen
at the times indicated. Ceramide was quantitated using DG kinase
(Escherichia coli). Results are expressed as the pmol/oocyte
and represent the mean of four samples.
Sphingomyelinase-induced GVBD Requires Protein
Synthesis
The requirement of protein synthesis during the
maturation of oocytes induced by progesterone is well known (Wasserman
and Masui, 1975). In order to determine whether protein synthesis is
required for sphingomyelinase-induced GVBD, oocytes were preincubated
with cycloheximide (100 µM) for 1 h prior to treatment of
cells with 0.5 units/ml sphingomyelinase for 5 min. Fig. 4shows
that cycloheximide blocked maturation of oocytes by both progesterone
and sphingomyelinase, suggesting that protein synthesis is required for
sphingomyelinase-induced maturation.
Figure 4:
Sphingomyelinase-induced GVBD requires
protein synthesis. Groups of 20 oocytes were treated with
sphingomyelinase (0.25 units/ml for 5 min, ) or progesterone (32
µM,
) or pretreated with cycloheximide (100
µM) and subsequently treated with sphingomyelinase (0.25
units/ml for 5 min,
) or progesterone (32 µM,
). Results are expressed as the percentage of total cells that
have undergone GVBD as measured by the appearance of a white spot on
the animal pole.
Microinjected Sphingolipids Induce GVBD
Varnold
and Smith have previously demonstrated that microinjection of
sphingosine was sufficient to induce GVBD. Thus, several sphingolipids
complexed to fatty acid-free BSA in a 1:1 molar ratio were
microinjected into oocytes at a final concentration of 40
µM. We also found that microinjected sphingosine was
sufficient to induce maturation (Fig. 5). Likewise,
phytosphingosine, a structurally similar sphingolipid, was also capable
of inducing maturation. The ability of these sphingolipids to trigger
meiosis was specific for the sphingoid backbone since stearylamine, a
long chain amine, at the same concentration had no effect. However,
since sphingosine can be acylated to yield ceramide, a bioactive lipid
(Hannun, 1994), we directly tested the ability of microinjected
ceramides to induce maturation of oocytes and found that both
C- and C
-ceramides were effective. In contrast,
erythro-N-acetyldihydrosphingosine and
threo-N-acetyldihydrosphingosine, analogs of ceramides that
lack a double bond or a 4`-OH, were inactive within the time course of
GVBD induced by short chain ceramides or sphingosine. In concordance
with previous results (Bielawska et al., 1993; Nakamura et
al., 1994), these data suggest that a double bond or a 4`-OH are
required for the biological activity of sphingolipids. Since ceramide
and sphingosine can be readily interconverted and the time course of
GVBD triggered by the injected sphingolipid-BSA complex may be affected
by exchange of the lipid off the BSA, it was necessary to determine
which lipid was the mediator of sphingolipid-induced GVBD. Thus, we
investigated the metabolism of microinjected sphingosine and ceramide.
Figure 5:
Time course of GVBD induced by
microinjection of sphingolipids. Groups of 20 oocytes were incubated
with progesterone (32 µM, ) or microinjected with BSA
(40 pmol) and subsequently treated with progesterone (32
µM,
), microinjected with phytosphingosine:BSA (40
pmol each, +), sphingosine:BSA (40 pmol each,
),
C
-ceramide:BSA (40 pmol each,
),
C
-ceramide:BSA (40 pmol each,
). Results are
expressed as a percentage of the total cells that have undergone GVBD
as measured by the appearance of a white spot on the animal
pole.
Sphingosine Is Metabolized to Ceramide by Xenopus
Oocytes
[H]Dihydrosphingosine and
C
-[
H]ceramide complexed to BSA was
microinjected into oocytes, and their metabolism followed.
Fig. 6A shows that [
H]sphingosine
is rapidly acylated to generate [
H]ceramide and
after an initial lag of 15 min to
[
H]sphingomyelin. Surprisingly,
C
-[
H]ceramide was converted to long
chain [
H]ceramides and
[
H]sphingomyelin without accumulation of
[
H]sphingosine. These data suggest that
sphingosine may be metabolized to ceramide prior to initiating
maturation. In contrast to Swiss 3T3 cells (Hauser et al.,
1994), the replacement of the N-acyl-linked hexanoic acid with
longer chain fatty acids demonstrate that the short chain analogs of
ceramides are readily metabolized by oocytes.
Figure 6:
Metabolism of sphingolipids by Xenopus oocytes. Oocytes were microinjected with
[H]dihydrosphingosine complexed to BSA or
C
-[
H]ceramide complexed to BSA.
Samples of oocytes were collected at the time intervals indicated, and
the total lipids were extracted and resolved by thin-layer
chromatography in a solvent system of chloroform/methanol/ammonium
hydroxide (65:35:8 (v/v/v)). Areas corresponding to labeled lipids were
scraped and quantitated by liquid scintillation counting. Results are
expressed as the total cpm in dihydrosphingosine (
), ceramide
(
), or sphingomyelin (
) (A) or
C
-ceramide (
), ceramide (
), sphingomyelin
(
) (B). These data are representative of one of three
independent experiments.
Fumonisin B
To further investigate if the acylation of sphingosine
to ceramide was necessary for induction of maturation, we used the
fungal metabolite, fumonisin BInhibits Sphingosine-induced
GVBD
. Fumonisin B
shares structural similarities with the sphingoid bases
sphinganine and sphingosine (Schroeder et al., 1994).
Fumonisin B
inhibits sphingosine N-acyltransferase
and causes accumulation of sphingoid bases (Wang et al.,
1991). Injection of fumonisin B
(40 µM final
concentration) prior to [
H]dihydrosphingosine:BSA
inhibits the acylation to [
H]ceramide with a
small accumulation of radiolabel, which comigrated with sphingosine
phosphate (Fig. 7A). We also found that fumonisin
B
blocked the conversion of
C
-[
H]ceramide to
H-labeled long-chain ceramide with an accumulation of
[
H]sphingosine (data not shown). These data
suggest that in oocytes, short chain analogs of ceramide are converted
to long chain ceramides by a sequence of reactions involving hydrolysis
by ceramidase followed by acylation of the resulting sphingosine.
Fumonisin B
blocks sphingosine but not progesterone-induced
GVBD (Fig. 7B). Likewise, fumonisin B
blocked the ability of microinjected C
-ceramide to
induce GVBD, suggesting that the conversion to long-chain ceramide
occurs prior to initiation of maturation (data not shown). Together,
these data demonstrate that sphingosine must first be acylated to
ceramide to induce GVBD.
Figure 7:
Effect of fumonisin B on
sphingosine-induced GVBD. The effect of fumonisin B
on the
metabolism of [
H]dihydrosphingosine is shown in
panelA. Oocytes were microinjected with
[
H]dihydrosphingosine complexed to BSA or
C
-[
H]ceramide complexed to BSA.
Samples of oocytes were collected at the time intervals indicated, and
the total lipids were extracted and resolved by thin-layer
chromatography in a solvent system of chloroform/methanol/ammonium
hydroxide (65:35:8 (v/v/v)). Areas corresponding to labeled lipids were
scraped and quantitated by liquid scintillation counting. Results are
expressed as the total cpm in dihydrosphingosine (
), ceramide
(
), or sphingosine phosphate (
). Groups of 20 oocytes were
microinjected with fumonisin B
(40 pmol) and incubated for
60 min. Cells were then treated with progesterone (
) or
microinjected with sphingosine (40 pmol) complexed to BSA (
).
Control cells were untreated cells incubated with progesterone (32
µM,
), or microinjected with sphingosine (40 pmol)
complexed to BSA (
). Results (panelB) are
expressed as a percentage of the total cells that have undergone GVBD
as measured by the appearance of a white spot on the animal pole. These
data are representative of one of three independent
experiments.
Progesterone Causes a Time-dependent Increase in Ceramide
Mass in Xenopus Oocytes
To investigate the physiological
relevance of ceramide-induced GVBD, experiments were conducted to
determine if ceramide levels change upon treatment of oocytes with
progesterone, the physiological inducer of maturation. Xenopus oocytes were treated with progesterone for various periods of
time, and the mass of ceramide was determined. Fig. 8, A and B, shows a time course of changes in ceramide mass
upon treatment of cells with 10 µg/ml progesterone (32
µM). A significant increase in the mass of ceramide was
observed within the first 5 min following progesterone treatment.
Within the first hour, the mass of ceramide increased from control
levels of approximately 250 to 500 pmol. Ceramide remained elevated at
2-3-fold over untreated cells for about 3 h. In order to
determine the source of the ceramide generated upon treatment of cells
with progesterone, the mass of sphingomyelin was measured. As shown in
Fig. 8C, progesterone treatment resulted in a 25%
decrease in the mass of sphingomyelin by 30-60 min and a 40%
decrease by 120-180 min. The immediate effect of progesterone on
the hydrolysis of sphingomyelin and the increase in the cellular
content of ceramide suggests that this lipid may serve as a messenger
molecule as seen in mammalian systems.
Figure 8:
Time course of ceramide formation in
Xenopus oocytes treated with progesterone. Groups of 20
oocytes were treated with progesterone (32 µM), and
samples of oocytes were collected at the time intervals indicated.
Ceramide was quantitated using DAG kinase (E. coli). Results
in panelA are expressed as pmol of ceramide/oocyte
for untreated cells () or progesterone-treated cells (
). The
corresponding autoradiograph of the thin-layer plate is shown in
panelB. The effect of progesterone on the mass of
sphingomyelin was determined as described under ``Experimental
Procedures,'' and the autoradiograph is shown in panelC. These data are representative of one of three
independent experiments.
Concentration Dependence of Progesterone on Ceramide
Levels
We next investigated the concentration dependence of
progesterone on ceramide levels. Oocytes were incubated with varying
concentrations of progesterone for 30 min, and ceramide levels were
determined. Fig. 9shows that 10 nM caused a significant
increase of ceramide mass, while 0.1 µM elicited a maximal
response at this time interval. This correlates with our observation
that 0.32 nM progesterone was not sufficient to induce
maturation while oocytes treated with 3.2 nM progesterone
underwent GVBD but with a slightly delayed response relative to higher
concentrations (data not shown).
Figure 9:
Ceramide formation in response to
progesterone. Groups of 20 oocytes were treated with varying
concentrations of progesterone for 30 min. Oocytes were collected and
assayed for ceramide using DAG kinase (E. coli). Results are
expressed as pmol of ceramide/nmol of sphingomyelin. Each data point
represents the average of duplicate samples ± the range. These
data are representative of one of two independent
experiments.
Progesterone Stimulates a
Mg
The mechanism of progesterone-induced
ceramide generation was investigated in oocytes. Therefore, assays were
performed to determine the activity of sphingomyelinases following
progesterone treatment. Fig. 10shows that in the presence of
Mg-dependent Neutral
Sphingomyelinase
, the specific activity of a neutral
sphingomyelinase was increased 3-4-fold following stimulation of
cells with progesterone, while the time course of stimulation was
similar to the increase in ceramide mass measured in lipid extracts.
This suggests that progesterone may directly or indirectly stimulate a
Mg
-dependent neutral sphingomyelinase, which in turn
would generate ceramide from sphingomyelin.
Figure 10:
Effect of progesterone on neutral
sphingomyelinase activity of Xenopus oocytes. Oocytes were
treated with progesterone (32 µM), and samples were
collected at the time intervals indicated. Cells were resuspended in
100 mM Tris-HCl, 5 mM EDTA, 0.1% Triton X-100, pH
7.4. Cells were disrupted by sonication and centrifuged at 10,000
g for 10 min. The supernatant was assayed for neutral
sphingomyelinase activity as described under ``Experimental
Procedures.'' Results are expressed as nmol/mg/h in the presence
of Mg
(
) or in the absence of Mg
(
). Each data point represents the average of duplicate
samples ± the range and are representative of one of three
independent experiments.
into Xenopus oocytes and assayed the onset of
maturation by examining for GVBD and H1 kinase activity. All three
enzymes were found to stimulate GVBD and H1 kinase activity. However,
these conditions may not be physiologically relevant since lipid second
messengers are transiently generated and the phospholipases were
present for extended periods of time (18 h). Additionally, it has been
our observation with microinjected phospholipases that the oocytes
degenerate, and a ``false GVBD'' phenotype is observed.
Therefore, in the present study we chose to treat defolliculated
oocytes exogenously with phospholipases for brief periods of time.
Under these conditions, phospholipase C and D treatment, even for 60
min, was not sufficient to induce maturation. However, in agreement
with previous reports (Varnold and Smith, 1990a, 1990b), treatment of
cells with sphingomyelinase (0.25 units/ml for 5 min) induced GVBD in
the absence of hormone that was morphologically indistinguishable from
progesterone-induced GVBD. Meiotic cell progression is driven, in part,
by p34
kinase. In oocytes arrested in prophase
of meiosis I, this activity, as measured as histone H1 phosphorylation,
is low and, in response to progesterone, increases, reaching peak
levels at the time of GVBD. Following meiosis I, the activity declines
but reaccumulates at the time of metaphase of meiosis II, where it
remains at high levels in the egg until the time of fertilization.
Thus, this activity is indicative of the cell's progression
through meiosis I and into meiosis II. Our results show a similar time
course and oscillation of H1 kinase activity of cells treated with
sphingomyelinase or progesterone thus demonstrating that
sphingomyelinase induces a normal maturation process.
inhibited sphingosine acylation and
blocked sphingosine-induced GVBD without affecting progesterone-induced
GVBD. Thus, these data suggest that an increase in the intracellular
content of ceramide is sufficient to overcome the prophase block and
allow reentry into the meiotic cell cycle.
(Okazaki
et al., 1989) and dexamethasone (Ramachandran et al.,
1990) have been reported to stimulate a sphingomyelin cycle in
mammalian cells. This involves the activation of neutral
sphingomyelinases and in some instances acidic sphingomyelinases
(Wiegmann et al., 1994) with a transient hydrolysis of plasma
membrane sphingomyelin to generate ceramide. It is proposed that the
majority of the ceramide is subsequently used for the resynthesis of
sphingomyelin in a reaction catalyzed by phosphatidylcholine:ceramide
phosphocholinetransferase (Merrill and Jones, 1990) and thus very
little would be converted to sphingosine. Addition of bacterial
sphingomyelinase or cell permeable analogs of ceramides to intact cells
have been used to demonstrate that ceramides mediate differentiation
(Okazaki et al., 1989), mitogenesis (Hauser et al.,
1994) and apoptosis (Obeid et al., 1993; Jarvis et
al., 1994). Ceramide is proposed to function as a second messenger
by activating a proline-directed protein kinase (Mathias et
al., 1991) and/or phosphatase (Dobrowsky and Hannun, 1992) of the
heterotrimeric family of protein phosphatases. Recently, ceramide was
shown to stimulate the activity of protein kinase C-
(Lozano,
1994). Since ceramide is a second messenger linked to the regulation of
mammalian cell growth and differentiation, we investigated if ceramide
was a physiologically relevant mediator of oocyte maturation. In this
respect, we found that progesterone treatment stimulates an increase in
the intracellular mass of ceramide in a time- and
concentration-dependent manner, and conclude that it is derived from
sphingomyelin. Additionally, we observed that progesterone stimulated a
Mg
-dependent neutral sphingomyelinase within the
first 2-5 min. Together, these results represent the first report
of the existence of a sphingomyelin cycle that may be functionally
important for reinitiation of the meiotic cell cycle triggered by
progesterone. The studies presented herein demonstrate a role for
ceramide in the maturation of oocytes and suggest that Xenopus oocytes are an excellent model for the study of ceramide-mediated
biology. Studies are currently being conducted to establish the
relative position of ceramide in the signal transduction pathways
leading to maturation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.