DEVELOPMENTAL
BIOLOGY
149, 206-212 (19%)
Activation
of Polyphosphoinositide Metabolism at Artificial Maturation of Pate//a vulgata Oocytes
BEATRICE BORG, GUY DE RENZIS, PATRICK PAYAN, AND BRIGITTE CIAPA’ Ldwratoire
de Physiologic Celluhire
et Comparhe, CNRS URA 651, UniversitS de Nice, Part Valroae, Nice Cedex, France Accepted September 19, 1991
The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella v-ulgata Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mMNH,CI, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of “PO, and rH]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol4,5-bisphosphate (PIP,). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol1,4,&trisphosphate (IP,) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Pate& oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP,, the messenger responsible for the Ca2+ signal at fertilizatiOn.
0 1992 Academic
Press,
Inc.
INTRODUCTION
Abundant literature has recently been published concerning the control of meiotic maturation. Most of this work deals with “cell cycle control proteins,” in an attempt to elucidate the nature of the cytoplasmic activity known as MPF (maturation promoting factor), which is essential for progression through the cell cycle (reviews by Smith, 1989; Guerrier et aL, 1990 a,b). However, the second messengers generated at the plasma membrane level and responsible for the production of active MPF are poorly defined. There is good evidence that a small decrease in CAMP could account for the onset of maturation in Xenopua oocytes (review by Smith, 1989). The role of calcium as second messenger remains controversial, however, as variations in intracellular free calcium concentration have been reported to occur (Moreau et al., 1978; Wasserman et aZ., 1980; Moreau et al, 1980; Witchel and Steinhardt, 1990) or not (Eisen and Reynolds, 1984; Robinson, 1985; Cork et a,!., 1987) after maturation of oocytes in various species. A few years ago, Guerrier et al. (1986a) proposed that meiosis reinitiation of Patella vulgata oocytes arrested at the germinal vesicle prophase stage proceeds in two steps. The first step, which leads to germinal vesicle breakdown (GVBD), chromosome condensation and metaphase-1 spindle formation, involves only a rise in ’ To whom correspondence should be addressed. 0012-1606/92 $3.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
206
intracellular pH. The second step, which releases the oocytes from the metaphase-1 block, depends on a rise in intracellular free calcium triggered by fertilization. In support of that model, Guerrier et al. (1986b) have found that prematurely fertilized GV-blocked oocytes did not activate unless they were treated further with weak bases such as ammonia to induce GVBD and formation of the metaphase-1 spindle. The mechanisms of maturation of this oocyte are therefore different from those in sea urchins, where fertilization takes place after completion of maturation and involves a transient calcium surge linked to a sustained alkalinization (reviews by Whitaker and Steinhardt, 1982; Epel 1989). In sea urchins, studies also strongly suggest that inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DAG), which come from the hydrolysis of phosphatidylinositol4,5-bisphosphate (PIP& (reviews by Berridge, 1987; Berridge and Irvine, 1989; Downes and MacPhee, 1990; Bansal and Majerus, 1990) are the crucial messengers at fertilization. IP, and DAG are produced after fertilization (Ciapa and Whitaker, 1986) and linked to an increased turnover of all polyphosphoinositides (PPI) (Turner et al, 1984; Kamel et ah, 1985; Swann et al., 1987). In sea urchin egg, these two messengers act by respectively releasing calcium from intracellular stores and increasing the cytosolic pH (pHi) by activating protein kinase C and thus the Na+/ H+ exchange (reviews by Swann et al., 1987; Ciapa et al., 1991). It was thus of interest to study the metabolism of polyphosphoinositides in P. vulgata eggs where the sig-
BORG ET AL.
PPI at Maturation
nals required for activating cellular processes during maturation and then fertilization occur separately. Some results have been reported concerning the involvement of PPI metabolism during oocyte maturation. However, it is unclear whether IP, plays any part in the oocyte maturation response, and variations in PIP2 are controversial as increases have been measured during meiotic reinitiation or after germinal vesicle breakdown (see review by Whitaker, 1989). Thus, the link between the polyphosphoinositide messenger system and oocyte maturation remains obscure. This paper shows that ammonia-induced maturation of P. vulgata oocytes causes a general increase of polypolyphosphoinositides synthesis, not only in PIP2 as in Xenopus oocytes (Le Peuch et ab, 1985), but also in PIP and PI. These events occur with the same time course as GVBD but seems to be a consequence of maturation rather than a condition necessary to elicit maturation. We suggest that the general increase in PPI turnover, concomitant with a significant increase in the stock of PPI, might ensure after maturation the substantial production of IP, which is thought to be responsible for the Ca2+ signal observed at the fertilization. MATERIAL
AND
METHODS
Oocyte Handling P. vulgata were collected from September to March in the vicinity of Roscoff and maintained at 12°C under a shallow layer of sea water. Prophase-blocked oocytes were obtained by cutting and agitating the gonads in artificial sea water (ASW: Marinemix Wiegant GMBH), pH 7.8. The remains of gonads were removed by passage through gauze, and oocyte suspensions were rinsed by successive decantations in ASW. Eggs concentrations were adjusted to 5% on a volume basis. All experiments reported here were performed on oocyte populations which did not present a percentage of spontaneous maturation higher than 5%. Meiosis reinitiation was performed in ASW (pH 8.2) at room temperature and triggered by adding to 5% oocyte suspensions NH&l from a 1 Mstock solution to give a final concentration of 10 mM. The appearance of GVBD, which typically occurs 10 min after ammonia addition, was observed by light microscopy. Oocyte populations presenting a percentage of maturation lower than 90% were rejected. Labeling of Polyphosphoinositi Polyphosphutes
and Inositol
A 20% suspension (v/v) of immature oocytes was incubated from 5 to 50 hr depending on experiments at
of Patella vulgata Oocytes
207
13°C in ASW (pH 7.8) containing an antibiotic (sulfadiazine 0.1%) in the presence of myo-[2-‘Hlinositol (10 &i/ml) or [52P]orthophosphate (100 &i/ml) (Amersham Inc.). Before maturation experiments, eggs were rinsed four times by decantation in cold ASW, pH 7.8. Extraction and Separation Polyphosphates
of Lipids
and Inositol
At different times following ammonia addition, 1 to 4 ml of the egg suspension was taken, centrifuged, and treated as follows. The supernatant was removed by aspiration and the pellet immediately resuspended in icecold trichloroacetic acid (TCA) (10% in distilled water) for 1 hr at 4°C. After centrifugation in an Eppendorf centrifuge, lipids were extracted from the pellet with chloroform/methanol/l1 M HCl (100/200/5 v/v) overnight at 4”C, and inositol polyphosphates isolated from the supernatant. Polyphosphoinositides were separated by chromatography on oxalate-treated silica gel thin-layer chromatography plates (T-6395 Sigma) in chloroform/methanol/acetone/acetic acid/water (40/13/15/12/g) (Jolles et al, 1981). After localization by autoradiography (films: X-OMAT Kodak), lipids were scraped from the TLC plates. In the case of [‘Hlinositol labeling experiments, TLC plates were previously sprayed with a scintillation mixture (0.4% PPO in P-methyl naphtalene made soluble in toluene) to permit detection by autoradiography. Radioactivity incorporated in the lipids was measured by scintillation counting. Phosphorus was measured by using the technique described by Rouser et al. (1970). Scraped spots were first treated with 11 N HCl(500 ,ul) for 10 min, at 18O”C, i.e., until total evaporation of HCl. They were then mineralized in 250 ~1 of perchloric acid (PCA) 70% for 1 hr at 180°C. Amounts of inorganic phosphorus were determined using an ammonium molybdate/ascorbic acid spectrophotometric assay (Rouser et ah, 1970). Protein content of the TCA pellet was determined according to the method described by Lowry et al (1951). RESULTS
Incorporation of “PO, and [‘Hynositol Polyphosph~nos~t~des of Immature
in Eggs
Figure 1A shows that the incorporation of both =PO, and [3H]inositol into immature eggs of Patek vulgata was linear and did not reach isotopic equilibrium even after 50 hr of incubation. Incubations were not prolonged more than 2 days, as after this time eggs cannot undergo normal maturation. The incorporations of 32P04 (Fig. 1B) and [‘Hlinositol (Fig. PI. , PIP. . and , - 10~, were measured in the three linids: *
208
DEVELOPMENTAL BIOLOGY 2000 r
whereas 32P0, accumulated more rapidly in PIP, than in PIP and PI. These differences arose because 32P0, was incorporated at three steps in the PPI cycle (PI, PIP, and PIP, synthesis) whereas inositol entered the PPI cycle only during PI synthesis.
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Effects of Artificial Maturation Incorporaticun into PPI 0
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;Y
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FIG. 1. Incorporation of “PO,, (@ and [‘Hlinositol (0) into immature eggs (A) and into polyphosphoinositides of immature eggs (B, C). (A) PI, (A) PIP, (0) PIP,. Eggs were labeled as described under Materials and Methods. Records presented are typical of three experiments. Data were obtained from the same oocyte population. (A) At different times following precursor addition, l-ml samples were taken and counted by liquid scintillation after washing the eggs with cold ASW. (B, C) Four milliliter samples were taken at different times following precursor addition, and lipids extracted and separated as described under Materials and Methods.
PIPB. The kinetics of incorporation of both radioactive precursors in all of the PPI were hyperbolic. This increasing accumulation can be explained if one considers that PPI are synthesized from “PO, and [3H]inositol, the intracellular specific radioactivity of which increased linearly (see Fig. 1A). The initial rates of incorporation of 32P04 or [3H]inositol in the different PPI indicated that [3H]inositol appeared first in PI, then in PIP, and finally in PIP,,
by Ammonia on “PO,
During a short (5-hr) incubation with 32P04, only polyphosphoinositides were labeled and clearly detectable on autoradiograms of TLC plates. After a longer incubation, phosphatidic acid and a trace of phosphatidylcholine were observed occasionally while phosphatidylethanolamines were not labeled. As these two lipids are the most abundant phospholipids in Patella eggs (results not shown), it is clear that PPI were the phospholipids with the fastest turnover in immature eggs. Figure 2 shows the alterations of the incorporation of 32P0, in the different PPI after maturation by 10 mM ammonia. In this set of experiments, eggs were incubated for 5 hr in the presence of the radiolabeled precursor before ammonia addition. We observed a high variability of incorporation of the tracer into immature eggs during the incubation period, even under identical experimental conditions. All results are thus displayed as the relative amount of 32P0., incorporated in the lipids of immature eggs at the end of the incubation period. As seen in Fig. 2, addition of ammonia induced an immediate increase in the incorporation of 32P0, into the three PPI. While the rate of 32P0, incorporation into PI remained stimulated for at least 50 min, it decreased for both PIP and PIP, about 10 min after addition of ammonia. An increased incorporation of the radioactive tracer into PPI might be the result of an acceleration of the turnover of the PPI cycle and/or a de rwvo synthesis of PPI. To clarify the issue a determination of the total phosphorus contained in the different PPI was carried out. Variations in Polyphosphoinositide Amounts at ArtQicial Maturation by Ammonia
As seen in Table 1, immature eggs contained 288.5 k 5.2 (n = 5) nmole of phosphorus/mg protein, which corresponds to a concentration of about 58 mM assuming a water space of 5 pl/mg protein. This result is in agreement with that obtained by X-ray microanalysis in our laboratory by I. Gillot on sea urchin eggs (personal communication). The amounts of phosphorus contained in total phospholipids, polyphosphoinositides, and in the three different PPI are also reported in Table 1. The concentrations of PIP and PIP, were of the same order of magnitude as those reported in sea urchin eggs by Turner et ccl. (1984). It must be noted that the amount of
PPI at Maturation
BORG ET AL.
209
of Patella mlgata Ooc@es TABLE 1 PHOSPHORUS DISTRIBUTION IN EGGS AND PHOSPHOLIPIDS AND CHEMICAL CONCENTRATIONS OF ALL PPI (B)
A Total Total PPI
B
oocytes phospholipids
288.5 + 5.2 57.6 f 0.9 9.0 + 1.1
Note. Results are the means expressed as nmole/mg prot8in. cantly different from zero.
I 10
0.9 0 1.6
I 20
I 30
I 40
I 50
rc
(A)
PI PIP PIP,
7.74 5 0.94 1.10 f 0.31 0.20 +- 0.13*
+ SEM of five experiments and are (*): For PIP, results are not signifi-
GVBD, which occurred within 10 min of ammonia addition, PPI synthesis took place, leading to a significant increase in the intracellular pools of these lipids. The situation occurring after GVBD is more complicated. At that time incorporation of %P04 into PIP and PIP, decreased (Fig. 2), whereas the phosphorus content remained constant (Fig. 3). This could be explained by a decrease in the specific radioactivity of ATP after completion of GVBD. In order to check this hypothesis, we stimulated maturation in [3H]inositol preloaded oocytes with ammonia. 1.6
-
1.5
-
A
0
10 TIME
20 (MIN)
AFTER
30 ADDITION
FIG. 2. Relative changes in 90, labeled (C) after ammonia stimulation. Eggs were ence of =PO, as described under Materials ment with 10 m&f ammonia. Each point ments. Results are expressed _tSEM and =PO, which was incorporated in the lipids end of the incubating period and arbitrarily
40
50
OF NH4CL
PI (A), PIP (B), and PIP, incubated for 5 hr in presand Methods before treatis the mean of six experirelative to the amount of of premature eggs at the taken as 1.0.
0
10
I .7
30
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B
1.6
-
1.5-
phosphorus contained in the PIP, is only just within the detection limit of the technique used. From our results it can be calculated that PI, PIP, and PIP, represent respectively 92.6, 6.6, and 0.8% of total PPI. Figure 3 shows that maturation by ammonia induced a 30 to 50% increase in both PI and PIP concentrations. Maximal concentrations of these lipids occurred 10 min after ammonia stimulation and were sustained for at least 50 min. Due to scatter in PIP, phosphorus measurements, it was impossible to detect any clear modification of the concentration of this lipid after artificial maturation. These results suggest that concomitant with
20
1.4
-
1.3
-
T I
I 10
0 TIME
(min)
I 20 AFTER
I 30 ADDITION
I 40 OF NHqCL
I 50
FIG. 3. Relative changes in chemical amounts of PI (A) and PIP (B) after ammonia stimulation. Each point corresponds to the mean HEM of six experiments. Results are relative to the chemical amount of each lipid which was measured in premature eggs at the end of the incubating period (Table 1) and arbitrarily taken as 1.0.
210 Changes in [‘Hynositol Polyphosphoinositides
DEVELOPMENTAL BIOLOGY
Incorporation at Artificial
VOLUME 149,19%
3r
in Maturation
In this set of experiments, oocytes were incubated for 48 hr in the presence of [3H]inositol. After this incubation, oocytes displayed the same rate of maturation as that observed in control oocytes, i.e., 90-100’S of GVBD occurring 10 min after ammonia addition. As pointed out for 32P04 incorporation, the accumulation of [3H]inositol in different batches of oocytes was variable. Results are thus presented as the relative amount of [3H]inositol present in the lipids of immature oocytes at the end of the incubation period. Figure 4 shows that stimulation of oocytes by ammonia triggered a biphasic increase in [3H]inositol incorporation into the three PPI. Maximal incorporation occurred during the first 10 min following ammonia treatment, i.e., until GVBD was completed. After this time, the incorporation of [3H]inositol in PPI remained linear over a period of 50 min. Table 2 shows that the rate of incorporation of [3H]inositol in the three PPI was about 45 times greater after GVBD in mature oocytes. This clearly demonstrates that ammonia stimulation also induced a large increase in the turnover of these lipids in addition to de novo PPI synthesis.
2.5
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DISCUSSION
The data presented in this paper demonstrate that meiosis reinitiation of Patella vulgata oocytes by ammonia triggers an increased synthesis of all polyphosphoinositides. The increase occurred not only with PIP2, as is the case in Xenopus oocytes matured with progesterone (Le Peuch et al, 1985), but also with PIP and PI. In agreement with the observations of Carrasco et al. (1990) in Xenopus oocytes, this work shows that maturation triggers increased turnover beside de nova synthesis of PPI. In particular, the incorporation of [3H]inosito1 into polyphosphoinositides remains stimulated even after GVBD is completed (Table 2). Investigations were carried out to see if the modifications in PPI turnover occurring after ammonia maturation led to a production of inositol polyphosphates. We observed that the stimulation of the PPI cycle did not cause an acute burst of IP, synthesis (results not shown) as generally observed in most cell activations (review by Berridge and Irvine, 1989), although it did produce a slow increase in the incorporation of [3H]inositol into the inositol polyphosphates measured (IP, IP,, IP,). This may be due to an increase in concentration of PIP and PIP,, leading to a mass action effect. The absence of IP, production at maturation of P. vulgata oocytes agrees with the conclusions of Guerrier et al (1986a,b) who proposed that GVBD in this oocyte depends mainly on a change in intracellular
0
10
20
30
40
50
TIME (mini AFTER ADDITION OF NH4CL FIG. 4. Alterations in [3H]inositol-labeled PPI after ammonia stimulation. Eggs were incubated for 50 hr with [‘Hlinositol as described under Materials and methods before the addition of 10 mM ammonia. Each point corresponds to the mean of six experiments. Results are expressed GEM and relative to the amount of PH]inositol which was incorporated in the lipids of premature eggs at the end of the incubating period and arbitrarily taken as 1.0.
pH, without any special requirement for calcium mobilization. The discrepancy between the kinetics of incorporations of %P04 and [3H]inositol after GVBD could be explained by a decrease in the specific radioactivity of ATP due to simultaneous decompartimentalization of inorganic phosphate and stimulation of ATP synthesis as a response of cell activation. The diminution of =PO, incorporation in PI would be less pronounced than in PIP and PIP, because PI is formed from CTP instead of ATP (review by Abdel-Latif, 1986). Finally, the fact that the rate of incorporation of 32P0, in PI is only eight times greater after GVBD than in immature eggs, com-
BORG ET AL.
TABLE EFFECT OF MATURATION [8H]I~~~~~ INCXXWRATION TELLA WLGATA &MXTES
Before maturation A PI PIP PIP,
3.5 2.3 2.1
PPI at Maturation
2
BY 10 mM AMMONIA ON THE RATE OF INTO POLYPHOSPHOINOSITIDES OF PA-
After maturation B 155 f 39 108 f 20 98 + 54
B/A 44 + 11 46? 4 44* 6
Note. Incorporation rates (A and B) were calculated as a percentage increase per hour, as follows: The amount of PH]inositol incorporated in the lipids of immature eggs at the end of the incubation period of 50 hr was arbitrarily taken as 1.0 and used as the reference value for all results. In immature eggs (one experiment was performed), the linear increase of [aH]inositol observed during the last 10 hr of incubation was expressed per hour and converted into an hourly percentage of the above reference value of 1.0. In mature eggs (six experiments were performed), incorporation of [‘H]inositol was compared between 10 and 30 min following ammonia addition. The percentage increase observed during this period of 20 min was again converted for 1 hr, as an hourly percentage of the reference value. The last column gives the B/A ratio.
pared to 45 times for [3H]inositol, reinforces this hypothesis. Another explanation involves the existence of heterogeneous pools of PPI, as demonstrated in cells such as platelets (Vickers and Mustard, 1986) or human erythrocytes (Gascard et a& 1990). The hormonal induction of meiosis reinitiation in P. vulgata is a two step process: a gonad stimulating substance of neural origin (GSS) induces the follicle cells surrounding the oocyte to produce a meiosis inducing substance (MIS) which acts directly on the oocytes (Guerrier et aL, 1990b). We have used ammonia for releasing P. vdgata oocytes from prophase I block as neither GSS nor MIS have yet been purified. The question can then be raised as to whether ammonia treatment mimics natural maturation. In P. vulgata oocytes it has been shown that ammonia addition stimulates a rise in intracellular pH (Guerrier et ak, 1986a), protein synthesis and phosphorylation (NQant and Guerrier, 1988), and germinal vesicle breakdown (Guerrier et cd., 198613) to levels equal to those observed when maturation is induced by addition of crude palleo-pedal ganglia extracts (Guerrier, personal communication, and personal observations). Moreover, 45 min after ammonia addition, eggs can be fertilized normally. These features encourage the belief that artificially maturated P. oocytescan be compared to oocytes matured naturally. The important question that emerges is whether the increase in PPI synthesis and its turnover on the one hand and maturation on the other hand are two linked events. Does one of these events induce the other? We found that defolliculating the oocytes (Guerrier et d,
of Patella vulgatu Oocytes
211
1986a) resulted in both an increase in the chemical amount of all PPI and in the incorporation of rH]inosito1 and =PO, in these lipids (data not shown). Stimulation of PPI synthesis and turnover induced by defolliculation were of the same order of magnitude as that observed after ammonia addition. However, such a treatment did not lead to GVBD and maturation of the defolliculated oocytes. This implies that the increase in PPI synthesis and turnover induced by ammonia is not sufficient to induce maturation but is a consequence of the meiosis reinitiation. The changes in the metabolism of PPI induced by the rise in intracellular pH might be related to stockpiling of PPI so that it is available for production of substantial amounts of IP,, the messenger responsible for the free calcium burst occurring at fertilization. It is odd that none of the second messengers which have been studied, for example polyphosphoinositide derivatives (our findings) and, as discussed by Witchel and Steinhardt (1990), calcium, CAMP, and arachidonic acid, are necessary and sufficient for maturation in oocytes. Many observations strongly suggest that a change in intracellular pH is the second messenger responsible for triggering maturation in P. vulgutu. The problem of the origin of this messenger is thus of importance. Data presented by Guerrier et al. (1986a) do not favor stimulation of a Ca+/H+ exchanger. Our observations that the phorbol ester TPA, which mimics DAG effect by activating protein kinase C (Nishizuka, 1986), can trigger maturation only if sodium is present in the external medium (unpublished results) suggest an activation of the Na+/H+ exchanger. As we did not measure any burst of IP, production, it is probable that no burst of production of DAG from PIP, occurs either. Another origin for DAG could be the hydrolysis of other phospholipids such as phosphatidylcholine (Exton, 1990). Experiments are now in progress to confirm this hypothesis. We thank Dr. R. House and M. Whitaker for correction of the manuscript and the “Commisariat a 1’Energie Atomique” (Department of Biology) for facilitating the purchase of radioactive products. We are grateful to Drs. M. Moreau, P. Guerrier, and F. Giraud for their comments and suggestions. REFERENCES Abdel-Latif, A. (1986). Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Phmmaco~ Rev. 38,227-273. Bansal, V. S., and Majerus, P. W. (1990). Phosphatidylinositol-derived precursors and signals. Ann. Rev. Physiol 52,41-67. Berridge, M. J. (1987). Inositols lipids and cell proliferation. Biochem. Biophys. Aeta 907,33-45. Berridge, M. J., and Irvine, R. F. (1989). Inositol phosphates and cell signalling. Nature 341, 197-205. Carrasco, D., Allende, C. C., and Allende, J. E. (1990). The incorporation of myo-inositol into phosphatidylinositol derivatives is stimu-
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lated during hormone induced meiotic maturation of amphibian oocytes. E3cp. Cell. Res. 191,313-318. Ciapa, B., and Whitaker, M. (1986). Two phases of inositol polyphosphate and diacyglycerol production at fertilization. FE&S. Leti, 195, 347-351. Ciapa, B., Borg, B., and Epel, D. (1991). Polyphosphoinosites, tyrosine kinase and sea urchin egg activation. In “Proceedings of the Seventh International Echinoderm Conference,” Sept. 9-14. Atami, Japan, in press Cork, R. J., Cicirelli, M. F., and Robinson, K. R. (1987). A rise in cytosolic calcium is not necessary for maturation of Xenopus luevis oocytes. Dev. Biol. 121,41-47. Downes, C. P., and MacPhee, C. H. (1990). myo-Inositol metabolites as cellular signals. Eur. J Biochem. 193, 1-18. Eisen, A., and Reynolds, G. (1984). Calcium transients during early development in single starfish oocytes. J. Cell Biol. 99,1878-1882. Epel, D. (1989). An ode to Edward Chambers: Linkage of transport, calcium and pH to sea urchin egg arousal at fertilization. In “Mechanism of Egg Activation” (R. Nuccitelli, G. N. Cherr, and W. H. Clark, Eds.), pp. 271-284. Plenum, New York/London. Exton, J. H. (1990). Signaling through phosphatidylcholine breakdown. J. Biol. Chem 265,1-4. Gascard, P., Journet, E., Sulpice, J. C., and Giraud, F. (1989). Functional heterogeneity of polyphosphoinostides in human erythrocytes. B&hem. J. 264,547-553. Guerrier, P., Brassart, M., David, C., and Moreau, M. (1986a). Sequential control of meiosis reinitiation by pH and Ca” in oocytes of the Prosobranch Mollusk Patella vu&a. Dev. Biol. 114,315-324. Guerrier, P., Colas, P., and Neant, I. (1990a). Meiosis reinitiation as a model system for the study of cell division and cell differentiation. Int. J. Den Biol. 34,93-109. Guerrier, P., Guerrier, C., Neant, I., and Moreau, M. (1986b). Germinal vesicle nucleoplasm and intracellular pH requirements for cytoplasmic maturity in oocytes of the prosobranch mollusk Patek vulgata. Dev. Biol. 116, 92-99. Guerrier, P., Neant, I., Colas, P., Dufresne, L., Saint Pierre, J., and Dube, F. (1990b). Protein synthesis and protein phosphorylation as regulators of MPF activity. In “Mechanisms of Fertilization,” NATO AS1 Series (B. Dale) (Ed.), Vol. H 45, pp. 79-100. SpringerVerlag, Berlin/Heidelberg. Jolles, J., Zwiers, H., Dekker, A., Wurtz, K. W. A., and Gispen, W. H. (1981). Corticotropin-(1.2.4)-tetracosapeptide affects protein phosphorylation metabolism in rat brain. B&hem. J. 194,283-291. Kamel, L. C., Bailey, J., Schoenbaum, L., and Kinsey, W. (1985). Phosphatidylinositol metabolism during fertilization in the sea urchin eggs. Lipids 20,350-356. Le Peuch, C. J., Picard, A., and Doree, M. (1985). Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs. FEBS Lett. 187, 61-64.
VOLUME
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Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, J. R. (1951). A method for estimation of protein. J. Biol Chem. 193,265-275. Moreau, M., Guerrier, P., Doree, M., and Ashley, C. C. (1978). Hormone-induced release of intracellular calcium triggers meiosis in starfish oocytes. Nature &m&m) 272,251-253. Moreau, M., Vilain, J. P., and Guerrier, P. (1980). Free calcium changes associated with hormone action in amphibian oocytes. Dev. Biol.78, 201-214. N&ant, I., and Guerrier, P. (1988). Meiosis reinitiation in the mollusc Patella wulgata. Regulation of MPF, CSF, and chromosome condensation activity by intracellular pH, protein synthesis and phosphorylation. Development 102.505-516. Nishizuka, Y. (1986). Studies and perspectives of protein kinase C. Science 233,305-312. Robinson, K. R. (1985). Maturation of Xenqpus oocytes is not accompanied by electrode-detectable calcium changes. Dev. Biol. 109, 504508. Rouser, G., Fleisher, S., and Yamamoto, A. (1970). Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lip& 5, 494-496. Schmell, E., and Lennarz, W. J. (1974). Phospholipids metabolism in the eggs and embryos of the sea urchin Arbacia punctulata. Biochemistry 13(20), 4114-4121. Smith, L. D. (1989). The induction of oocyte maturation: transmembrane signalling events and regulation of the cell cycle. Develop merit 10’7.685-699. Swann, K., Ciapa, B., and Whitaker, M. (1987). Cellular messenger and sea urchin eggs activation. In “Molecular Biology of Invertebrate” (D. 0. Connors, Ed.), 45-69. A. R. Liss, New York. Turner, P. R., Sheetz, M. P., and Jaffe, L. A. (1984). Fertilization increases the polyphosphoinositides content of sea urchin eggs. Nature 310,414-415. Vickers, J. D., and Mustard, J. F. (1986). The phosphoinositides exist in multiple metabolic polls in rabbit platelets. B&hem. J. 238, 411417. Wasserman, W. J., Pinto, L. H., O’Connor, C. M., and Smith, L. D. (1980). Progesterone induces a rapid increase in [Ca’+], of Xenopus laevis oocytes. Proc. Nat1 Ad. Sci USA 77,1534-1536. Whitaker, M. (1989). Phosphoinositide second messengers in eggs and oocytes. In “Inositol Lipids in the Cell Signalling” (R. H. Michell, A. H. Drummond, and C. P. Downes, Eds.), pp 459-483. Academic Press, New York. Whitaker, M., and Steinhardt, R. A. (1982). Ionic regulation of egg activation. Q. Rev. Biophys. 15, 593-666. Witchel, H. H., and Steinhardt, R. A. (1990). I-Methyladenine can consistently induce a fura-detectable transient calcium increase which is neither necessary nor sufficient for maturation in oocytes of the starfish Asterina miniatu. Dev. Biol. 141.393-398.
BIOLOGY
149, 206-212 (19%)
Activation
of Polyphosphoinositide Metabolism at Artificial Maturation of Pate//a vulgata Oocytes
BEATRICE BORG, GUY DE RENZIS, PATRICK PAYAN, AND BRIGITTE CIAPA’ Ldwratoire
de Physiologic Celluhire
et Comparhe, CNRS URA 651, UniversitS de Nice, Part Valroae, Nice Cedex, France Accepted September 19, 1991
The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella v-ulgata Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mMNH,CI, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of “PO, and rH]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol4,5-bisphosphate (PIP,). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol1,4,&trisphosphate (IP,) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Pate& oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP,, the messenger responsible for the Ca2+ signal at fertilizatiOn.
0 1992 Academic
Press,
Inc.
INTRODUCTION
Abundant literature has recently been published concerning the control of meiotic maturation. Most of this work deals with “cell cycle control proteins,” in an attempt to elucidate the nature of the cytoplasmic activity known as MPF (maturation promoting factor), which is essential for progression through the cell cycle (reviews by Smith, 1989; Guerrier et aL, 1990 a,b). However, the second messengers generated at the plasma membrane level and responsible for the production of active MPF are poorly defined. There is good evidence that a small decrease in CAMP could account for the onset of maturation in Xenopua oocytes (review by Smith, 1989). The role of calcium as second messenger remains controversial, however, as variations in intracellular free calcium concentration have been reported to occur (Moreau et al., 1978; Wasserman et aZ., 1980; Moreau et al, 1980; Witchel and Steinhardt, 1990) or not (Eisen and Reynolds, 1984; Robinson, 1985; Cork et a,!., 1987) after maturation of oocytes in various species. A few years ago, Guerrier et al. (1986a) proposed that meiosis reinitiation of Patella vulgata oocytes arrested at the germinal vesicle prophase stage proceeds in two steps. The first step, which leads to germinal vesicle breakdown (GVBD), chromosome condensation and metaphase-1 spindle formation, involves only a rise in ’ To whom correspondence should be addressed. 0012-1606/92 $3.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
206
intracellular pH. The second step, which releases the oocytes from the metaphase-1 block, depends on a rise in intracellular free calcium triggered by fertilization. In support of that model, Guerrier et al. (1986b) have found that prematurely fertilized GV-blocked oocytes did not activate unless they were treated further with weak bases such as ammonia to induce GVBD and formation of the metaphase-1 spindle. The mechanisms of maturation of this oocyte are therefore different from those in sea urchins, where fertilization takes place after completion of maturation and involves a transient calcium surge linked to a sustained alkalinization (reviews by Whitaker and Steinhardt, 1982; Epel 1989). In sea urchins, studies also strongly suggest that inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DAG), which come from the hydrolysis of phosphatidylinositol4,5-bisphosphate (PIP& (reviews by Berridge, 1987; Berridge and Irvine, 1989; Downes and MacPhee, 1990; Bansal and Majerus, 1990) are the crucial messengers at fertilization. IP, and DAG are produced after fertilization (Ciapa and Whitaker, 1986) and linked to an increased turnover of all polyphosphoinositides (PPI) (Turner et al, 1984; Kamel et ah, 1985; Swann et al., 1987). In sea urchin egg, these two messengers act by respectively releasing calcium from intracellular stores and increasing the cytosolic pH (pHi) by activating protein kinase C and thus the Na+/ H+ exchange (reviews by Swann et al., 1987; Ciapa et al., 1991). It was thus of interest to study the metabolism of polyphosphoinositides in P. vulgata eggs where the sig-
BORG ET AL.
PPI at Maturation
nals required for activating cellular processes during maturation and then fertilization occur separately. Some results have been reported concerning the involvement of PPI metabolism during oocyte maturation. However, it is unclear whether IP, plays any part in the oocyte maturation response, and variations in PIP2 are controversial as increases have been measured during meiotic reinitiation or after germinal vesicle breakdown (see review by Whitaker, 1989). Thus, the link between the polyphosphoinositide messenger system and oocyte maturation remains obscure. This paper shows that ammonia-induced maturation of P. vulgata oocytes causes a general increase of polypolyphosphoinositides synthesis, not only in PIP2 as in Xenopus oocytes (Le Peuch et ab, 1985), but also in PIP and PI. These events occur with the same time course as GVBD but seems to be a consequence of maturation rather than a condition necessary to elicit maturation. We suggest that the general increase in PPI turnover, concomitant with a significant increase in the stock of PPI, might ensure after maturation the substantial production of IP, which is thought to be responsible for the Ca2+ signal observed at the fertilization. MATERIAL
AND
METHODS
Oocyte Handling P. vulgata were collected from September to March in the vicinity of Roscoff and maintained at 12°C under a shallow layer of sea water. Prophase-blocked oocytes were obtained by cutting and agitating the gonads in artificial sea water (ASW: Marinemix Wiegant GMBH), pH 7.8. The remains of gonads were removed by passage through gauze, and oocyte suspensions were rinsed by successive decantations in ASW. Eggs concentrations were adjusted to 5% on a volume basis. All experiments reported here were performed on oocyte populations which did not present a percentage of spontaneous maturation higher than 5%. Meiosis reinitiation was performed in ASW (pH 8.2) at room temperature and triggered by adding to 5% oocyte suspensions NH&l from a 1 Mstock solution to give a final concentration of 10 mM. The appearance of GVBD, which typically occurs 10 min after ammonia addition, was observed by light microscopy. Oocyte populations presenting a percentage of maturation lower than 90% were rejected. Labeling of Polyphosphoinositi Polyphosphutes
and Inositol
A 20% suspension (v/v) of immature oocytes was incubated from 5 to 50 hr depending on experiments at
of Patella vulgata Oocytes
207
13°C in ASW (pH 7.8) containing an antibiotic (sulfadiazine 0.1%) in the presence of myo-[2-‘Hlinositol (10 &i/ml) or [52P]orthophosphate (100 &i/ml) (Amersham Inc.). Before maturation experiments, eggs were rinsed four times by decantation in cold ASW, pH 7.8. Extraction and Separation Polyphosphates
of Lipids
and Inositol
At different times following ammonia addition, 1 to 4 ml of the egg suspension was taken, centrifuged, and treated as follows. The supernatant was removed by aspiration and the pellet immediately resuspended in icecold trichloroacetic acid (TCA) (10% in distilled water) for 1 hr at 4°C. After centrifugation in an Eppendorf centrifuge, lipids were extracted from the pellet with chloroform/methanol/l1 M HCl (100/200/5 v/v) overnight at 4”C, and inositol polyphosphates isolated from the supernatant. Polyphosphoinositides were separated by chromatography on oxalate-treated silica gel thin-layer chromatography plates (T-6395 Sigma) in chloroform/methanol/acetone/acetic acid/water (40/13/15/12/g) (Jolles et al, 1981). After localization by autoradiography (films: X-OMAT Kodak), lipids were scraped from the TLC plates. In the case of [‘Hlinositol labeling experiments, TLC plates were previously sprayed with a scintillation mixture (0.4% PPO in P-methyl naphtalene made soluble in toluene) to permit detection by autoradiography. Radioactivity incorporated in the lipids was measured by scintillation counting. Phosphorus was measured by using the technique described by Rouser et al. (1970). Scraped spots were first treated with 11 N HCl(500 ,ul) for 10 min, at 18O”C, i.e., until total evaporation of HCl. They were then mineralized in 250 ~1 of perchloric acid (PCA) 70% for 1 hr at 180°C. Amounts of inorganic phosphorus were determined using an ammonium molybdate/ascorbic acid spectrophotometric assay (Rouser et ah, 1970). Protein content of the TCA pellet was determined according to the method described by Lowry et al (1951). RESULTS
Incorporation of “PO, and [‘Hynositol Polyphosph~nos~t~des of Immature
in Eggs
Figure 1A shows that the incorporation of both =PO, and [3H]inositol into immature eggs of Patek vulgata was linear and did not reach isotopic equilibrium even after 50 hr of incubation. Incubations were not prolonged more than 2 days, as after this time eggs cannot undergo normal maturation. The incorporations of 32P04 (Fig. 1B) and [‘Hlinositol (Fig. PI. , PIP. . and , - 10~, were measured in the three linids: *
208
DEVELOPMENTAL BIOLOGY 2000 r
whereas 32P0, accumulated more rapidly in PIP, than in PIP and PI. These differences arose because 32P0, was incorporated at three steps in the PPI cycle (PI, PIP, and PIP, synthesis) whereas inositol entered the PPI cycle only during PI synthesis.
0
A
VOLUME 14% 19%
1500 -
Effects of Artificial Maturation Incorporaticun into PPI 0
10
20
30
40
50
4000
B z
#
3000
:: 2% g $ 2000 F,E dL 02 y 1000 0
;Y
k PI
b
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0
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20
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40
r c
I
50
/
P
3000 2000
t
1000
0
0 E
10
20 TIME
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40
50
(hours)
FIG. 1. Incorporation of “PO,, (@ and [‘Hlinositol (0) into immature eggs (A) and into polyphosphoinositides of immature eggs (B, C). (A) PI, (A) PIP, (0) PIP,. Eggs were labeled as described under Materials and Methods. Records presented are typical of three experiments. Data were obtained from the same oocyte population. (A) At different times following precursor addition, l-ml samples were taken and counted by liquid scintillation after washing the eggs with cold ASW. (B, C) Four milliliter samples were taken at different times following precursor addition, and lipids extracted and separated as described under Materials and Methods.
PIPB. The kinetics of incorporation of both radioactive precursors in all of the PPI were hyperbolic. This increasing accumulation can be explained if one considers that PPI are synthesized from “PO, and [3H]inositol, the intracellular specific radioactivity of which increased linearly (see Fig. 1A). The initial rates of incorporation of 32P04 or [3H]inositol in the different PPI indicated that [3H]inositol appeared first in PI, then in PIP, and finally in PIP,,
by Ammonia on “PO,
During a short (5-hr) incubation with 32P04, only polyphosphoinositides were labeled and clearly detectable on autoradiograms of TLC plates. After a longer incubation, phosphatidic acid and a trace of phosphatidylcholine were observed occasionally while phosphatidylethanolamines were not labeled. As these two lipids are the most abundant phospholipids in Patella eggs (results not shown), it is clear that PPI were the phospholipids with the fastest turnover in immature eggs. Figure 2 shows the alterations of the incorporation of 32P0, in the different PPI after maturation by 10 mM ammonia. In this set of experiments, eggs were incubated for 5 hr in the presence of the radiolabeled precursor before ammonia addition. We observed a high variability of incorporation of the tracer into immature eggs during the incubation period, even under identical experimental conditions. All results are thus displayed as the relative amount of 32P0., incorporated in the lipids of immature eggs at the end of the incubation period. As seen in Fig. 2, addition of ammonia induced an immediate increase in the incorporation of 32P0, into the three PPI. While the rate of 32P0, incorporation into PI remained stimulated for at least 50 min, it decreased for both PIP and PIP, about 10 min after addition of ammonia. An increased incorporation of the radioactive tracer into PPI might be the result of an acceleration of the turnover of the PPI cycle and/or a de rwvo synthesis of PPI. To clarify the issue a determination of the total phosphorus contained in the different PPI was carried out. Variations in Polyphosphoinositide Amounts at ArtQicial Maturation by Ammonia
As seen in Table 1, immature eggs contained 288.5 k 5.2 (n = 5) nmole of phosphorus/mg protein, which corresponds to a concentration of about 58 mM assuming a water space of 5 pl/mg protein. This result is in agreement with that obtained by X-ray microanalysis in our laboratory by I. Gillot on sea urchin eggs (personal communication). The amounts of phosphorus contained in total phospholipids, polyphosphoinositides, and in the three different PPI are also reported in Table 1. The concentrations of PIP and PIP, were of the same order of magnitude as those reported in sea urchin eggs by Turner et ccl. (1984). It must be noted that the amount of
PPI at Maturation
BORG ET AL.
209
of Patella mlgata Ooc@es TABLE 1 PHOSPHORUS DISTRIBUTION IN EGGS AND PHOSPHOLIPIDS AND CHEMICAL CONCENTRATIONS OF ALL PPI (B)
A Total Total PPI
B
oocytes phospholipids
288.5 + 5.2 57.6 f 0.9 9.0 + 1.1
Note. Results are the means expressed as nmole/mg prot8in. cantly different from zero.
I 10
0.9 0 1.6
I 20
I 30
I 40
I 50
rc
(A)
PI PIP PIP,
7.74 5 0.94 1.10 f 0.31 0.20 +- 0.13*
+ SEM of five experiments and are (*): For PIP, results are not signifi-
GVBD, which occurred within 10 min of ammonia addition, PPI synthesis took place, leading to a significant increase in the intracellular pools of these lipids. The situation occurring after GVBD is more complicated. At that time incorporation of %P04 into PIP and PIP, decreased (Fig. 2), whereas the phosphorus content remained constant (Fig. 3). This could be explained by a decrease in the specific radioactivity of ATP after completion of GVBD. In order to check this hypothesis, we stimulated maturation in [3H]inositol preloaded oocytes with ammonia. 1.6
-
1.5
-
A
0
10 TIME
20 (MIN)
AFTER
30 ADDITION
FIG. 2. Relative changes in 90, labeled (C) after ammonia stimulation. Eggs were ence of =PO, as described under Materials ment with 10 m&f ammonia. Each point ments. Results are expressed _tSEM and =PO, which was incorporated in the lipids end of the incubating period and arbitrarily
40
50
OF NH4CL
PI (A), PIP (B), and PIP, incubated for 5 hr in presand Methods before treatis the mean of six experirelative to the amount of of premature eggs at the taken as 1.0.
0
10
I .7
30
40
50
B
1.6
-
1.5-
phosphorus contained in the PIP, is only just within the detection limit of the technique used. From our results it can be calculated that PI, PIP, and PIP, represent respectively 92.6, 6.6, and 0.8% of total PPI. Figure 3 shows that maturation by ammonia induced a 30 to 50% increase in both PI and PIP concentrations. Maximal concentrations of these lipids occurred 10 min after ammonia stimulation and were sustained for at least 50 min. Due to scatter in PIP, phosphorus measurements, it was impossible to detect any clear modification of the concentration of this lipid after artificial maturation. These results suggest that concomitant with
20
1.4
-
1.3
-
T I
I 10
0 TIME
(min)
I 20 AFTER
I 30 ADDITION
I 40 OF NHqCL
I 50
FIG. 3. Relative changes in chemical amounts of PI (A) and PIP (B) after ammonia stimulation. Each point corresponds to the mean HEM of six experiments. Results are relative to the chemical amount of each lipid which was measured in premature eggs at the end of the incubating period (Table 1) and arbitrarily taken as 1.0.
210 Changes in [‘Hynositol Polyphosphoinositides
DEVELOPMENTAL BIOLOGY
Incorporation at Artificial
VOLUME 149,19%
3r
in Maturation
In this set of experiments, oocytes were incubated for 48 hr in the presence of [3H]inositol. After this incubation, oocytes displayed the same rate of maturation as that observed in control oocytes, i.e., 90-100’S of GVBD occurring 10 min after ammonia addition. As pointed out for 32P04 incorporation, the accumulation of [3H]inositol in different batches of oocytes was variable. Results are thus presented as the relative amount of [3H]inositol present in the lipids of immature oocytes at the end of the incubation period. Figure 4 shows that stimulation of oocytes by ammonia triggered a biphasic increase in [3H]inositol incorporation into the three PPI. Maximal incorporation occurred during the first 10 min following ammonia treatment, i.e., until GVBD was completed. After this time, the incorporation of [3H]inositol in PPI remained linear over a period of 50 min. Table 2 shows that the rate of incorporation of [3H]inositol in the three PPI was about 45 times greater after GVBD in mature oocytes. This clearly demonstrates that ammonia stimulation also induced a large increase in the turnover of these lipids in addition to de novo PPI synthesis.
2.5
A
-
4r
0
B
10
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30
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50
DISCUSSION
The data presented in this paper demonstrate that meiosis reinitiation of Patella vulgata oocytes by ammonia triggers an increased synthesis of all polyphosphoinositides. The increase occurred not only with PIP2, as is the case in Xenopus oocytes matured with progesterone (Le Peuch et al, 1985), but also with PIP and PI. In agreement with the observations of Carrasco et al. (1990) in Xenopus oocytes, this work shows that maturation triggers increased turnover beside de nova synthesis of PPI. In particular, the incorporation of [3H]inosito1 into polyphosphoinositides remains stimulated even after GVBD is completed (Table 2). Investigations were carried out to see if the modifications in PPI turnover occurring after ammonia maturation led to a production of inositol polyphosphates. We observed that the stimulation of the PPI cycle did not cause an acute burst of IP, synthesis (results not shown) as generally observed in most cell activations (review by Berridge and Irvine, 1989), although it did produce a slow increase in the incorporation of [3H]inositol into the inositol polyphosphates measured (IP, IP,, IP,). This may be due to an increase in concentration of PIP and PIP,, leading to a mass action effect. The absence of IP, production at maturation of P. vulgata oocytes agrees with the conclusions of Guerrier et al (1986a,b) who proposed that GVBD in this oocyte depends mainly on a change in intracellular
0
10
20
30
40
50
TIME (mini AFTER ADDITION OF NH4CL FIG. 4. Alterations in [3H]inositol-labeled PPI after ammonia stimulation. Eggs were incubated for 50 hr with [‘Hlinositol as described under Materials and methods before the addition of 10 mM ammonia. Each point corresponds to the mean of six experiments. Results are expressed GEM and relative to the amount of PH]inositol which was incorporated in the lipids of premature eggs at the end of the incubating period and arbitrarily taken as 1.0.
pH, without any special requirement for calcium mobilization. The discrepancy between the kinetics of incorporations of %P04 and [3H]inositol after GVBD could be explained by a decrease in the specific radioactivity of ATP due to simultaneous decompartimentalization of inorganic phosphate and stimulation of ATP synthesis as a response of cell activation. The diminution of =PO, incorporation in PI would be less pronounced than in PIP and PIP, because PI is formed from CTP instead of ATP (review by Abdel-Latif, 1986). Finally, the fact that the rate of incorporation of 32P0, in PI is only eight times greater after GVBD than in immature eggs, com-
BORG ET AL.
TABLE EFFECT OF MATURATION [8H]I~~~~~ INCXXWRATION TELLA WLGATA &MXTES
Before maturation A PI PIP PIP,
3.5 2.3 2.1
PPI at Maturation
2
BY 10 mM AMMONIA ON THE RATE OF INTO POLYPHOSPHOINOSITIDES OF PA-
After maturation B 155 f 39 108 f 20 98 + 54
B/A 44 + 11 46? 4 44* 6
Note. Incorporation rates (A and B) were calculated as a percentage increase per hour, as follows: The amount of PH]inositol incorporated in the lipids of immature eggs at the end of the incubation period of 50 hr was arbitrarily taken as 1.0 and used as the reference value for all results. In immature eggs (one experiment was performed), the linear increase of [aH]inositol observed during the last 10 hr of incubation was expressed per hour and converted into an hourly percentage of the above reference value of 1.0. In mature eggs (six experiments were performed), incorporation of [‘H]inositol was compared between 10 and 30 min following ammonia addition. The percentage increase observed during this period of 20 min was again converted for 1 hr, as an hourly percentage of the reference value. The last column gives the B/A ratio.
pared to 45 times for [3H]inositol, reinforces this hypothesis. Another explanation involves the existence of heterogeneous pools of PPI, as demonstrated in cells such as platelets (Vickers and Mustard, 1986) or human erythrocytes (Gascard et a& 1990). The hormonal induction of meiosis reinitiation in P. vulgata is a two step process: a gonad stimulating substance of neural origin (GSS) induces the follicle cells surrounding the oocyte to produce a meiosis inducing substance (MIS) which acts directly on the oocytes (Guerrier et aL, 1990b). We have used ammonia for releasing P. vdgata oocytes from prophase I block as neither GSS nor MIS have yet been purified. The question can then be raised as to whether ammonia treatment mimics natural maturation. In P. vulgata oocytes it has been shown that ammonia addition stimulates a rise in intracellular pH (Guerrier et ak, 1986a), protein synthesis and phosphorylation (NQant and Guerrier, 1988), and germinal vesicle breakdown (Guerrier et cd., 198613) to levels equal to those observed when maturation is induced by addition of crude palleo-pedal ganglia extracts (Guerrier, personal communication, and personal observations). Moreover, 45 min after ammonia addition, eggs can be fertilized normally. These features encourage the belief that artificially maturated P. oocytescan be compared to oocytes matured naturally. The important question that emerges is whether the increase in PPI synthesis and its turnover on the one hand and maturation on the other hand are two linked events. Does one of these events induce the other? We found that defolliculating the oocytes (Guerrier et d,
of Patella vulgatu Oocytes
211
1986a) resulted in both an increase in the chemical amount of all PPI and in the incorporation of rH]inosito1 and =PO, in these lipids (data not shown). Stimulation of PPI synthesis and turnover induced by defolliculation were of the same order of magnitude as that observed after ammonia addition. However, such a treatment did not lead to GVBD and maturation of the defolliculated oocytes. This implies that the increase in PPI synthesis and turnover induced by ammonia is not sufficient to induce maturation but is a consequence of the meiosis reinitiation. The changes in the metabolism of PPI induced by the rise in intracellular pH might be related to stockpiling of PPI so that it is available for production of substantial amounts of IP,, the messenger responsible for the free calcium burst occurring at fertilization. It is odd that none of the second messengers which have been studied, for example polyphosphoinositide derivatives (our findings) and, as discussed by Witchel and Steinhardt (1990), calcium, CAMP, and arachidonic acid, are necessary and sufficient for maturation in oocytes. Many observations strongly suggest that a change in intracellular pH is the second messenger responsible for triggering maturation in P. vulgutu. The problem of the origin of this messenger is thus of importance. Data presented by Guerrier et al. (1986a) do not favor stimulation of a Ca+/H+ exchanger. Our observations that the phorbol ester TPA, which mimics DAG effect by activating protein kinase C (Nishizuka, 1986), can trigger maturation only if sodium is present in the external medium (unpublished results) suggest an activation of the Na+/H+ exchanger. As we did not measure any burst of IP, production, it is probable that no burst of production of DAG from PIP, occurs either. Another origin for DAG could be the hydrolysis of other phospholipids such as phosphatidylcholine (Exton, 1990). Experiments are now in progress to confirm this hypothesis. We thank Dr. R. House and M. Whitaker for correction of the manuscript and the “Commisariat a 1’Energie Atomique” (Department of Biology) for facilitating the purchase of radioactive products. We are grateful to Drs. M. Moreau, P. Guerrier, and F. Giraud for their comments and suggestions. REFERENCES Abdel-Latif, A. (1986). Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Phmmaco~ Rev. 38,227-273. Bansal, V. S., and Majerus, P. W. (1990). Phosphatidylinositol-derived precursors and signals. Ann. Rev. Physiol 52,41-67. Berridge, M. J. (1987). Inositols lipids and cell proliferation. Biochem. Biophys. Aeta 907,33-45. Berridge, M. J., and Irvine, R. F. (1989). Inositol phosphates and cell signalling. Nature 341, 197-205. Carrasco, D., Allende, C. C., and Allende, J. E. (1990). The incorporation of myo-inositol into phosphatidylinositol derivatives is stimu-
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lated during hormone induced meiotic maturation of amphibian oocytes. E3cp. Cell. Res. 191,313-318. Ciapa, B., and Whitaker, M. (1986). Two phases of inositol polyphosphate and diacyglycerol production at fertilization. FE&S. Leti, 195, 347-351. Ciapa, B., Borg, B., and Epel, D. (1991). Polyphosphoinosites, tyrosine kinase and sea urchin egg activation. In “Proceedings of the Seventh International Echinoderm Conference,” Sept. 9-14. Atami, Japan, in press Cork, R. J., Cicirelli, M. F., and Robinson, K. R. (1987). A rise in cytosolic calcium is not necessary for maturation of Xenopus luevis oocytes. Dev. Biol. 121,41-47. Downes, C. P., and MacPhee, C. H. (1990). myo-Inositol metabolites as cellular signals. Eur. J Biochem. 193, 1-18. Eisen, A., and Reynolds, G. (1984). Calcium transients during early development in single starfish oocytes. J. Cell Biol. 99,1878-1882. Epel, D. (1989). An ode to Edward Chambers: Linkage of transport, calcium and pH to sea urchin egg arousal at fertilization. In “Mechanism of Egg Activation” (R. Nuccitelli, G. N. Cherr, and W. H. Clark, Eds.), pp. 271-284. Plenum, New York/London. Exton, J. H. (1990). Signaling through phosphatidylcholine breakdown. J. Biol. Chem 265,1-4. Gascard, P., Journet, E., Sulpice, J. C., and Giraud, F. (1989). Functional heterogeneity of polyphosphoinostides in human erythrocytes. B&hem. J. 264,547-553. Guerrier, P., Brassart, M., David, C., and Moreau, M. (1986a). Sequential control of meiosis reinitiation by pH and Ca” in oocytes of the Prosobranch Mollusk Patella vu&a. Dev. Biol. 114,315-324. Guerrier, P., Colas, P., and Neant, I. (1990a). Meiosis reinitiation as a model system for the study of cell division and cell differentiation. Int. J. Den Biol. 34,93-109. Guerrier, P., Guerrier, C., Neant, I., and Moreau, M. (1986b). Germinal vesicle nucleoplasm and intracellular pH requirements for cytoplasmic maturity in oocytes of the prosobranch mollusk Patek vulgata. Dev. Biol. 116, 92-99. Guerrier, P., Neant, I., Colas, P., Dufresne, L., Saint Pierre, J., and Dube, F. (1990b). Protein synthesis and protein phosphorylation as regulators of MPF activity. In “Mechanisms of Fertilization,” NATO AS1 Series (B. Dale) (Ed.), Vol. H 45, pp. 79-100. SpringerVerlag, Berlin/Heidelberg. Jolles, J., Zwiers, H., Dekker, A., Wurtz, K. W. A., and Gispen, W. H. (1981). Corticotropin-(1.2.4)-tetracosapeptide affects protein phosphorylation metabolism in rat brain. B&hem. J. 194,283-291. Kamel, L. C., Bailey, J., Schoenbaum, L., and Kinsey, W. (1985). Phosphatidylinositol metabolism during fertilization in the sea urchin eggs. Lipids 20,350-356. Le Peuch, C. J., Picard, A., and Doree, M. (1985). Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs. FEBS Lett. 187, 61-64.
VOLUME
149,1992
Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, J. R. (1951). A method for estimation of protein. J. Biol Chem. 193,265-275. Moreau, M., Guerrier, P., Doree, M., and Ashley, C. C. (1978). Hormone-induced release of intracellular calcium triggers meiosis in starfish oocytes. Nature &m&m) 272,251-253. Moreau, M., Vilain, J. P., and Guerrier, P. (1980). Free calcium changes associated with hormone action in amphibian oocytes. Dev. Biol.78, 201-214. N&ant, I., and Guerrier, P. (1988). Meiosis reinitiation in the mollusc Patella wulgata. Regulation of MPF, CSF, and chromosome condensation activity by intracellular pH, protein synthesis and phosphorylation. Development 102.505-516. Nishizuka, Y. (1986). Studies and perspectives of protein kinase C. Science 233,305-312. Robinson, K. R. (1985). Maturation of Xenqpus oocytes is not accompanied by electrode-detectable calcium changes. Dev. Biol. 109, 504508. Rouser, G., Fleisher, S., and Yamamoto, A. (1970). Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lip& 5, 494-496. Schmell, E., and Lennarz, W. J. (1974). Phospholipids metabolism in the eggs and embryos of the sea urchin Arbacia punctulata. Biochemistry 13(20), 4114-4121. Smith, L. D. (1989). The induction of oocyte maturation: transmembrane signalling events and regulation of the cell cycle. Develop merit 10’7.685-699. Swann, K., Ciapa, B., and Whitaker, M. (1987). Cellular messenger and sea urchin eggs activation. In “Molecular Biology of Invertebrate” (D. 0. Connors, Ed.), 45-69. A. R. Liss, New York. Turner, P. R., Sheetz, M. P., and Jaffe, L. A. (1984). Fertilization increases the polyphosphoinositides content of sea urchin eggs. Nature 310,414-415. Vickers, J. D., and Mustard, J. F. (1986). The phosphoinositides exist in multiple metabolic polls in rabbit platelets. B&hem. J. 238, 411417. Wasserman, W. J., Pinto, L. H., O’Connor, C. M., and Smith, L. D. (1980). Progesterone induces a rapid increase in [Ca’+], of Xenopus laevis oocytes. Proc. Nat1 Ad. Sci USA 77,1534-1536. Whitaker, M. (1989). Phosphoinositide second messengers in eggs and oocytes. In “Inositol Lipids in the Cell Signalling” (R. H. Michell, A. H. Drummond, and C. P. Downes, Eds.), pp 459-483. Academic Press, New York. Whitaker, M., and Steinhardt, R. A. (1982). Ionic regulation of egg activation. Q. Rev. Biophys. 15, 593-666. Witchel, H. H., and Steinhardt, R. A. (1990). I-Methyladenine can consistently induce a fura-detectable transient calcium increase which is neither necessary nor sufficient for maturation in oocytes of the starfish Asterina miniatu. Dev. Biol. 141.393-398.
