©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ceramide Triggers Meiotic Cell Cycle Progression in Xenopus Oocytes
A POTENTIAL MEDIATOR OF PROGESTERONE-INDUCED MATURATION (*)

Jay C. Strum , Katherine I. Swenson , J. Eric Turner , Robert M. Bell (§)

From the (1) Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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 NaSO, 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 NaVO (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:HO (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 HO bath. Reactions were terminated by the addition of 3 ml of methanol/chloroform (2:1 (v/v)) and 0.7 ml of HO. Phases were separated by the subsequent addition of 1.0 ml of chloroform and 1.0 ml of HO. 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.


RESULTS

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 BInhibits Sphingosine-induced GVBD

To further investigate if the acylation of sphingosine to ceramide was necessary for induction of maturation, we used the fungal metabolite, fumonisin B. 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-dependent Neutral Sphingomyelinase

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, 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.




DISCUSSION

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 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.

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 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.

A variety of agonists including the steroids 1,25-dihydroxyvitamin D (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.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant DK20205 (to R. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Glaxo Inc., Five Moore Dr., Research Triangle Park, NC 27709. Tel.: 919-990-6144; Fax: 919-990-6150.

The abbreviations used are: DAG, diacylglycerol; GVBD, germinal vesicle breakdown; BSA, bovine serum albumin.


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