Early stimulation of phospholipid methylation in Xenopus oocytes by progesterone

('ell Dii[7k,rentiation. 16 (1985) 35 41 Elsevier Scientific Publishers Ireland, Ltd.

35

C D F 00245

Early stimulation of phospholipid methylation in Xenopus oocytes by progesterone F r a n c o i s G o d e a u 1,., T e r u k o I s h i z a k a 2 and S.S. K o i d e i / 77w Population ('otmcil, 1230 York ,4 ce, New )'or/,, N )" 10021; and : Johns Hop/,ins L,'n#rer~'io', School o/' Medicine at the (,ood Samaritan tto.vlma/, Baltimore. MI) 21239. U.S./t. (Accepted 17 September 1984)

Progesterone induced a transient increase in the incorporation of

[3H]methyl groups

into phospholipids of

Xenopus oocytes followed by a rise in 4SCa2+ uptake. Phospholipid methylation reached a maximum as early as 15 s after progesterone treatment and returned to basal level within 2 min. Steroids inactive in promoting oocyte maturation were less effective in affecting phospholipid methylation. Methyltransferase inhibitors, 3-deaza-SIBA, SIBA, and Sinefungin, inhibited progesterone-activated stimulation of phospholipid methylation, calcium uptake and meiotic maturation. Phospholipid methylation is the earliest detectable biochemical event occurring in oocytes after exposure to progesterone followed b) calcium influx and leading to germinal vesicle dissolution. oocyte maturation: Xenopus: phospholipid-methylation: progesterone

Introduction Full-grown amphibian oocytes re-enter cell cycle and undergo the first meiotic division (maturation) in vitro upon exposure to progesterone (Masui, 1967: Maller and Krebs, 1980). The maturation process in the oocyte is initiated by the interaction of the hormone with the plasma membrane (Godeau et al., 1978b) and the message is relayed mtracellularly by cAMP and Ca 2+ through a cascade of events that remains to be clarified (Mailer and Krebs, 1980). Current evidence supports the thesis that cAMP may act as a 'negative effector" of progesterone action (Godeau et al., 1978a, 1981). Progesterone may inhibit the mere* Prc~em address: Dana-Farber Cancer Institute. Boston. MA 02115. L.S.A. (on leave from INSERM, Paris, France).

brane-bound adenylate cyclase (Sadler and Mailer, 1981), thereby affecting a drop in cAMP levels within 2 5 rain (Schorderet-Slatkine et al., 1982). Adenylate cyclase inhibition would induce a decrease in the steady-state level of cAMP-dependent protein phosphorylation of a phosphoprotein(s) which has been postulated to mediate the prophase block (Mailer and Krebs, 1977). On tile other hand, calcium ion may act as the 'positive effector' of progesterone action (Baulieu et al., 1978). Exposure to calcium ionophore A23187 (Wasserman and Masui, 1975), calcium iontophoresis (Moreau et al., 1976), and microinjection of a calcium-calmodulin complex (Wasserman and Smith, 1981) induce meiotic maturation in the absence of the hormone. Moreover, a rapid increase in intracellular free calcium concentration takes place within 1 2 nlin after treatment with

0045-6039/85/,'$03.30 ' 1985 Elsexier Scientific Publishers Ireland, Lid.

36 progesterone (Wasserman et al., 1980). The present study was undertaken to investigate the involvement of phospholipid methylation in the early changes of free calcium concentration brought about by progesterone in Xenopus oocytes. Evidence will be presented showing that the incorporation of [3H]methyl groups into phospholipids increases within 15-30 s after exposure of stage VI Xenopus oocytes to progesterone, followed by a rise in aSCa2+ uptake.

Materials and Methods

Materials and preparation of oocytes Adult Xenopus laevis females were purchased from the South African Snake Farm, Fish Hoek (Cape Province, South Africa) and maintained in the laboratory. Stage VI (Dumont, 1972) oocytes were prepared by extensive collagenase digestion of pieces of ovary surgically removed from the animal as described elsewhere (Eppig and Dumont, 1976). Stage VI oocytes were individually selected under a stereomicroscope and kept in sterile modified Barth's solution HEPES (MBSH) (Ford and Gurdon, 1977) containing antibiotics at room temperature. 45Ca 2+ (16.37 mCi/mg) and [methyl3H]methionine (15 Ci/mmol) were purchased from New England Nuclear Corp. 5'-Deoxy-5'(isobutylthio)-3-deaza-adenosine (3-deaza-SIBA) was synthetized as described (Hirata et al., 1978) and dissolved in absolute ethanol. Sinefungin was purchased from Calbiochem and dissolved in injection medium (88 mM NaC1/10 mM Hepes, pH 7.6). 5'-Deoxy-5'(isobutylthio)adenosine (SIBA) was purchased from Sigma and dissolved in dimethylsulfoxide (DMSO). All steroids were obtained from Sigma and dissolved in absolute ethanol. All solutions were diluted with sterile MBSH before use. All phospholipid standards, phosphatidylethanolamine (PE), phosphatidyl-Nmonomethylethanolamine (PME), phosphatidylN,N-dimethylethanolamine (PDE), phosphatidylcholine (PC) and lysophosphatidylcholine (LyPC) were obtained from Grand Island Biochemical Co.

Determination of phospholipid methvlation A group of 500 oocytes was metabolically labelled with 6.6/,tool of L-[~H]methylmethionine in 0.5 ml of MBSH for 1 h at room temperature. The oocytes were washed once with MBSH, and 10 washed oocytes were placed in individual tubes containing 40/,tl of MBSH. To each tube 10/21 of progesterone at a final concentration of 10 ~M were added. The reaction was stopped by the addition of 1 ml of ice-cold 10% trichloroacetic acid (TCA) containing 10 mM L-methionme. Oocytes were disrupted by three freeze-thawing cycles. The disrupted oocytes were centrifuged at 1 8 0 0 0 × g for 10 rain at 4°C. The pellet was washed with 10% TCA. The pellet was extracted with 3 ml of chloroform methanol mixture (2 : 1). The organic phase was washed with 1.5 ml of 0.1 M KC1 in 50% methanol. One ml aliquot of the chloroform phase was transferred to a scintillation vial, evaporated to dryness and processed for liquid scintillation spectroscopy (Hirata et al., 1978).

Analysis o[ methylated phospholipid~ A group of 500 oocytes was labeled with 20 /,tool L-[methyl-3H]methionine in 1 ml. To each tube, 40 washed oocytes were placed and challenged with 10 >M of progesterone. At 15 s after the challenge, the reaction was stopped by the addition of 10% TCA containing 10 mM Lmethionine, and phospholipids were extracted. To avoid oxidation of phospholipids, the chloroform phase was dried under a stream of nitrogen gas and the residue was dissolved in 50 >1 of chloroform/methanol (2 : 1. v/v). The samples were applied on a silica gel G plate (Uniplate, Analtech Inc., Newark, DE), and chromatograms were developed in a solvent system of chloroform/ acetone/methanol/acetic acid/water (5 : 2:1 : 1 : 0.5, v / v ) (Hirata et al.. 1978). Analysis by two-dimensional TL(" of silicone first plate was per-

formed using chloroform/nlethanol/arnmonia ( 6 5 : 2 5 : 4 , v/v) first and the above n-fixture as solvent for the second dimension.

Determination ~4 calcium influx Groups of 10 oocytes were placed in a tube containing 40/~1 of MBSH. To each tube 50 ~l of

37

MBSH solution containing 45Ca2~ (2 t t C i / m l ) and progesterone to give a final concentration of 10 ttM were added. The reaction was terminated by adding 100 btl of a solution containing 120 mM N a C I / 5 mM KC1/20 mM E D T A / 2 5 mM TrisHC1, pH 7.6/0.2 m g / m l BSA. Oocytes were layered on top of a solution of 14.7% metrizoate in MBSH in a microfuge tube. The tubes were centrifuged at 10000 × g for 1 min. The cell pellet at the bottom of the tube was collected by cutting off the tip. One ml of 1% Triton X-100 was added, and the cells were disrupted by sonication. Radioactivity in 100 ttl of the solution was measured in a scintillation spectrometer. 45Ca2+ uptake was expressed as the difference in radioactive counts between the progesterone-treated oocytes and untreated control cells.

Maturation experiments Groups of 50 oocytes (stage V1) were incubated in a petri dish containing 3 ml of MBSH. Maturation was induced by continuous exposure to 10 /,M progesterone (Godeau et al., 1978b) and monitored by the appearance of a white dot in the center of the animal pole, signifying the breakdown of the germinal vesicle (GVBD). Oocytes were fixed at the end of the incubation period in 2.5% glutaraldehyde and dissected to identify the GV. 3-Deaza-SIBA and SIBA were added to the incubation medium containing progesterone. Since cells are relatively impermeable to Sinefungin (Robert-Gero et al., 1979), this compound was microinjected into the oocyte. Each oocyte was injected with 50 nl of a concentrated solution of Sinefungin dissolved in the injection medium (88 mM N a C I / 1 0 mM Hepes buffer, pH 7.6), transferred to MBSH containing the inhibitor and immediately exposed to 10 ttM progesterone. In some experiments oocytes were microinjected with Sinefungm and placed in the incubation medium without the compound. The vehicles (1% acetone or lq: DMSO) per se did not influence the maturation process. results

When oocytes preincubated with [methylH]methionine were treated with progesterone, the

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Fig. 1. Kinetics of [~H]methyl incorporation and 45Ca 2+ uptake after exposure of Xenopus oocytes to 10 ttM progesterone. [~H]Methyl group incorporation into oocyte's phospholipids (e e) was determined as described under experimental procedures. Duplicate groups of oocytes (10 cells/tube) were incubated for varying duration in the presence of progesterone. Four experiments of the same design gave similar results. 4~Ca2+ upake (O O) was determined at varying incubation periods. Each point represents the mean of duplicate assays.

incorporation of [3H]methyl groups into the phospholipid fraction of the oocytes increased significantly (Fig. 1). A 2-4-fold increase in the amount of radioactivity incorporated into phospholipids was observed. Maximal incorporation occurred TABLE 1 Steroid specificity' of [3H]methyl incorporation into oocyte phospholipids

Xenopus

Steroids

Incorporation * (%)

None Progesterone. 0.1 kt M Progesterone. 1 ttM Progesterone. 10 tt M 19-Nortestosterone, 0.1 10,uM Estrone, 0.1 10/*M 17/%Estradiol, 10/*M Hvdrocortisone 10 tzM

100 95 160 158 98 103 87 108 93 100

* Oocvtcs were labelled vdth [methyl-3H]methionine as described in Methods. Groups of 10 oocytes were treated with the indicated steroid. The incubation was stopped after 15 s bv addithm of ice-cold 10~ TCA containing 10 mM unlabelled methionme. [~ H]methyl group incorporation was determined as described in Methods.

38 TABLE II Incorporation of [~ H]methyl into phospholipids after stimulation with progesterone :~ Phospholipids (cpm/40 cells) h

Control Progesterone Progesterone plus 1-deaza-S1BA ~

PE

PA

PC

PI

PDE

LyPC

PS

PM E

181 230 173

127 124 144

318 1 748 228

163 362 236

163 304 133

244 468

192 278 176

162 698 128

232

~ Oocytes (40 cells/tube) preincubated with [methyl-~H]methionine (20/*M/ml) were challenged with 10/~M progesterone. At 15 s after the challenge, phospholipids were extracted from the ceils and analyzed by two-dimensional TLC on silica G plate using chloroform/methanol/ammonia (65 : 25:4), and chloroform/acetone/methanol/acetic acid/water (5 : 2 : 1 : 1:0.5) as solvents. Values are means of two separate experiments. ~' Values are means of two separate experiments. Abbreviations: PE = phosphatidylethanolamine; PA phosphatidic acid: PI phosphatidylinositol: PDE = phosphatidyl-N,N-dimethylethanolamine; PC = phosphatidylcholine: I.y-PC lysophosphatidylcholine; PS = phosphatidylserine: PME = phosphatidyl-N-monomethylethanolamine. Oocytes were preincubated with 1 mM 3-deaza-SIBA at room temperature for 1 h and then challenged with progesterone.

15 s after the addition of progesterone. The level remained elevated up to 30 s and gradually declined to the baseline within 5 min, The effective concentrations of progesterone in stimulating [3H]methyl group incorporation into phospholipids were 1-10 /*M (Table I). Estrone and 17fiestradiol showed no stimulation of phospholipid methylation (Table I). To identify the products of phospholipid meth-

ylation, oocytes labeled with L-[methyl--'H]methionine were challenged with 10 /,M progesterone. At 15 s after challenge, the phospholipids were extracted from the cells and analyzed by thin layer chromatography on silica G plate. As shown in Table II, the incorporation of radiolabeled methyl into PME, PDE, and PC was markedly enhanced upon exposure to progesterone. A small, hut significant increase in

TABLE 111 Effect of methyltransferases inhibitors on progesterone-induced phospholipid methylation and calcium uptake in Xenopus oocytes Compound

3 Deaza-SlBA

Sinefungin *

Concentration (raM)

[3H]Methyl incorporation (% control)

45Ca2 ~ Uptake

Control 0.1 1.0 Control 0.1 1.0

(a) 100 56 30 100 82 40

(c) 100 73 19 100 n.d. 36

(b) 97 56 23 n.d. n.d. n.d.

(% control)

* Microinjected into oocyte: n.d. = not determined. After metabolic labelling of oocytes with [methyl -3 H]methionine, [~ H]melhyl group incorporation was determined as described m Materials and Methods at 15 s (a) or 30 s (b) after progesterone addition. Control value designated as 100'74 represents the increment of [~H]methyl group incorporation brought about by progesterone in the absence of inhibitor over unstimulated oocytes at the indicated time. Thus 100% is equivalent to 473 c p m / 1 0 ooeytes. 45Ca 2~ uptake (c) was determined as described in Materials and Methods 3 rain after progesterone addition. Similarly, the control value indicated as 100% is the increment of calcium uptake induced by progesterone in the absence of inhibitor as compared to stimulated oocytes. 3-l)eaza-SIBA was added to the incubation medium at the indicated concentration, and oocytes were incubated ill the presence of the drug for 1 h before addition of progesterone. Sinefungin was diluted to the appropriate concentration with the injectiona medium (see Methods) and microinjected into oocytes to give the estimated intracellular concentration. The volume of each oocyte was estimated to be 1 jttl. The time lapse between the last injection and the challenge with progesterone was 10 rain.

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Time (hr.) Fig. 2. Effect of methyhransferase inhibitors on meiotic maturation induced by progesterone. (a) Oocytes were preincubated for 1 h in the presence of 3-deaza-SIBA (1 raM, • i" 0.1 mM, ~ . . . . . . A) or l mM SIBA (A. . . . . . A) or in medium alone (0 0). After the addition of progesterone (10/~M) the time course of GVBD was determined as described in Materials and Methods. (b) Oocytes were injected with Sinefungin (1 mM • n: 0.1 mM A. . . . . . ex) or with medium alone (O 0) and placed in MBSH containing the same concentration of Sinefungin and immediately challenged with 10 p.M progesterone. Maturation was scored as described in Materials and Methods.

used, the rise in calcium uptake stimulated by progesterone was inhibited in a dose-dependent manner (Table III). Oocytes were microinjected with Sinefungin and incubated in a medium containing the c o m p o u n d at the same concentration. Sinefungin at 1 m M blocked the increase in calcium influx induced with progesterone. The degree of inhibition of calcium uptake was strongly correlated with that of phospholipid methylation brought about by inhibitors of methyltransferases at the concentrations used (r = 0.96). Experiments were also carried out to determine whether methyltransferase inhibitors could affect the biological response of oocyte to progesterone. As shown in Fig. 2, methyltransferases inhibitors also inhibited progesterone-induced maturation in a dose-dependent manner. Sinefungin was more effective than S-isobutyl-3-deazaadenosine. These inhibitors at the concentration used partially blocked progesterone response while SIBA at 1 mM, as previously reported (Schorderet-Slatkine et al., 1981), totally inhibited G V B D response to progesterone.

Discussion lysophosphatidylcholine was also detected 15 s after the stimulation. When a portion of oocytes were preincubated with 1 m M 3-deaza-SIBA for 1 h and then challenged with progesterone, the incorporation of [~H]methyl groups into methylated phospholipids was markedly inhibited. Uptake of 45Ca2+ by oocytes after progesterone stimulation was also examined. As shown in Fig. 1, a significant increase in calcium uptake was detected 30 s after the addition of the steroid. It reached a plateau after 1 min and remained elevated for at least 5 min. The incorporation of radioactivity increased from 10 to 30 cpm per oocyte during the initial minute after progesterone treatment. The kinetic studies show that phospholipid methylation is stimulated by progesterone prior to the increase in calcium uptake. To determine possible involvement of phospholipid methylation in Ca 2+ influx the effect of inhibitors of S-adenosyl-L-methionine dependent methyltransferases on 45Ca2+ influx was examined. When S-isobutyl-3-deazaadenosine was

The present data show that the steroid hormone, progesterone, stimulates phospholipid methylation in Xenopus oocytes. The effective dose in activating phospholipid methylation corresponds to the concentration required to demonstrate its biological effect (Sadler and Mailer, 1982). Progesterone at a concentration of 0.2 /~M was reported to induce G V B D in only 50% of oocytes (Sadler and Maller, 1982). In the present study the h o r m o n e at 0.1 mM did not stimulate phospholipid methylation (Table I) nor affected significant G V B D . Furthermore, progesterone was most active in stimulating phospholipid methylation while other steroids inactive in inducing oocyte maturation or inhibiting adenylate cyclase activity were less effective (Finidori-Lepicard et al., 1981). It is noteworthy that the stimulation of phospholipid methylation precedes the increase in calcium uptake. This observed temporal sequence together with the inhibition of calcium uptake by methyltransferase inhibitors strongly suggests that phospholipid

40 methylation may be linked to the opening of calcium channels. Collagenase w,'as used to isolate the oocytes. Following this enzyme treatment the preparation may contain oocyte follicle cells attached to oocytes. The observed 45Ca2' uptake may be by the follicle cells rather than the oocytes (O'Connor et al., 1977). The effect of methyltransferase inhibitors on the overall biological response of the oocytes to progesterone is difficult to interpret since these drugs may interfere with maturation at more than one locus, i,e., cAMP metabolism (Zimmerman et al., 1980). The observed increase in methylation of PI (Table II) is of interest since hormones that stimulate the metabolism of Pl also accelerate the entry of external Ca 2+ (Fain and Berridge, 1979a,b). Moreover, changes in the lipid components of the membrane may influence permeability to Ca 2~ and the conversion of PI to deacylglycerol may regulate Ca 2+ gating (Michell, 1975). Our present results also show that phospholipid methylation may be the earliest detectable biochemical event following exposure to progesterone. Decrease in intracellular cAMP was demonstrated 1 2 min after hormone addition by SchorderetSlatkine et al. (1982). Moreover, treatment with SIBA affected an mcrease in cAMP level (Schorderet-Slatkine et al., 1982). These findings suggest that stimulation of phospholipid methylation and adenylate cyclase activity may be associated events. Phospholipid methylation occurs earlier than the transient increase in intracellular free calcium reported by Wasserman et al. (40 60 s) (Wasserman et al., 1980). Our time course data of 45Ca2+ uptake agreed with those obtained by these investigators using microinjected Aequorin assay system. Both studies support the thesis that the observed increase in intracellular free calcium may have resulted from opening of calcium channels in the oocyte membrane subsequent to membrane fluidity changes resulting from methylation and translocation of phospholipids (Hirata and Axelrod, 1978a,b; lshizaka et al., 1981). Moreover, lshizaka et al. (1981) demonstrated that a strong correlation exists between phospholipid methylation and opening of calcium chalmels in rat mast cells stimulated with anti-lgE receptor antibodies. The present findings support a similar conclu-

sion in the case of progesterone stimulation of Xenopus oocytes where calcium ions are believed to act as positive effectors of progesterone action (Baulieu et al., 1978: Mailer and Krebs, 1980: Wasserman and Smith, 1981). The similarity of the timing of phospholipid methylation and calcium uptake in the two unrelated biological systems is striking considering the differences in cell type, optimal temperature (20 vs. 37°C), and ultimate biological responses, i.e., mast cell degranulation vs. resumption of cell division after release of a cell cycle blockade. They also differ considerably in their timing (few minutes vs. several hours). In addition, it was shown recently that bridging of IgE receptors in mast cell plasma membranes activated adenylate cyclase (Ishizaka et al., 1981), whereas in Xenopus oocytes, progesterone inhibited this enzyme (Sadler and Mailer, 1981). The reason for this opposing effect is not clear. In any event, the demonstration of a progesterone-stimulated phospholipid methylation occurring 15 s after addition of the hormone provides additional evidence that this steroid acts directly on oocyte membrane as proposed earlier (Godeau et al., 1978b, Baulieu et al., 1978). This notion is further supported by the recent demonstration of a progesterone-binding protein in oocyte plasma membrane (Sadler and Maller, 1982: Blondeau and Baulieu, 1984). Our results also suggest that phospholipid methylation may trigger calcium influx and lead to germinal vesicle breakdown.

Acknowledgemenls This study was supported in part by grant No, ROI-HD-13184 from the N I C H H D , All0060 from N I A I D and GA PS 8310 from the Rockefeller Foundation. The authors express their appreciation to Mr. Alan R. Sterk for his excellent technical assistance. This article is publication No. 586 from the O'Neill Laboratories at the Good Samaritan Hospital.

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