Hormonal control of meiosis in starfish: stimulation of protein phosphorylation induced by 1-methyladenine

Molecular and Cellular i+rdocrinology,

0 Elsevier/North-Holland

7 (1977) 137-150 Scientific Publishers, Ltd.

HORMONAL CONTROL OF MEIOSIS IN STARFISH: PROTEIN PHOSPHORYLATION

Pierre CUERRIER, Station Biologique,

STIMULATION

OF

INDUCED BY l-METHYLADENINE

Marc MOREAU and Marcel DOREE

2921 I Roscoff,

France

Received 9 September 1976; accepted 26 November 1976

Phosphorylation of proteins is strongly stimulated as early as 5 min after addition of 1-methyladenine, the hormone which triggers meiosis reinitiation in starfish, to prophase arrested oocytes of Marthasterias glacialis. Both biochemical and biological responses to the hormone are almost identical with respect to concentration dependence, kinetic characteristics, reversibility and sensibility to various inhibitors, which suggests that protein phosphorylation is a prerequisite for triggering the release of meiosis inhibition. Protein phosphorylation is stimulated more rapidly and extensively in the cortex of the oocyte than in the endoplasm, suggesting that phosphorylation of some cortical proteins might be a major and very early event in meiosis reinitiation. Keywords:

meiosis; protein phosphorylation;

l-methyladenine;

starfish

In starfish, full-grown oocytes are arrested at the prophase of meiosis. Meiosis is reinitiated by a ‘relay hormone’, 1-methyladenine (1 -MA) *, produced by the follicle cells under the influence of a peptide hormone of neural origin (Kanatani, 1973). It has been suggested that the first step in l-MA activation is its interaction with specific ‘receptors’ located on the cell membrane (Kanatani and Hiramoto, 1970; Kanatani and Shirai, 1971; Do&e and Guerrier, 1975; DorCe et al., 1976). Other authors (Hirai et al., 1971) showed that the interaction of l-MA with the membrane brought about cytoplasmic maturation which is expressed by the acquisition of fertilizability. More recently, Kishimoto and Kanatani (1976) reported that the changes in the cortical region of 1 -MA stimulated oocytes produce an unidentified cytoplasmic factor (MPF) which ,induces germinal vesicle breakdown (GVBD) leading to completion of oocyte maturation. The nature of biochemical changes that must occur after 1-MA addition has been poorly investigated. The only results reported to date are by Kishimoto et al. (1976) who showed that a significant increase in the amount of protein-SH occurs in the oocyte cortex 10 min after l-MA addition, and by Guerrier et al. (1975) who * Abbreviations used inOhis paper: l-MA, 1-methyladenine; GVBD, germinal vesicle breakdown; DTT, dithiothreitol. 137

MPF, maturation promoting factor;

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reported that protein kinase activity of whole oocyte homogenates was increased as early as 5 min after 1 -MA addition. In this paper some kinetic evidence is presented which shows that, following the addition of I-MA, protein phosphorylation is stimulated in both the cortical and endoplasmic region of starfish oocytes. The relationship between this stimulation of protein phosphorylation and the reinitiation of the meiosis program is also discussed.

MATERIALS

AND METHODS

Chemicals 13*P] P04H2Na, 2 Ci/mM, was purchased from C.E.A., France, and adenosine~‘-[Y-~*P] triphosphate, 2 Ci/mM, from Amersham, England. Unlabelled biochemical compounds were obtained from Sigma, U.S.A. Leupeptin was a gift of Drs. Aoyagi and Umezawa, University of Tokyo. Collecting isolated oocytes Oocytes were prepared free of follicle cells and suspended water as described previously (DorCe and Guerrier, 1975).

in calcium-free

sea

In vivo studies of 32P incorporation into oocytes About 200 /.&i of [32P]P04H2Na were injected into 100 ml of oocyte suspension (about 3 X lo4 oocytes/ml) which was maintained homogeneous by gentle stirring. After exposure to 32P for 1 h, oocytes were collected by gentle centrifugation, washed three times with Ca*+-free sea water and finally resuspended in this medium. After addition of 1-MA, 1 ml aliquots of the cell suspension were collected as a function of time and injected into 15% ice-cold TCA containing 10 mM PO:-. Precipitation of proteins was allowed to occur overnight. In the first experiments protein phosphate was determined according to the Schmidt-Thannhauser technique (1945). We found, however, no difference with the following simplified technique which was then used routinely. TCA precipitates were collected by centrifugation, washed three times with ice-cold 5% TCA followed by one wash with 100% EtOH. 3 ml 0.5 N NaOH were added and the precipitate was heated for 10 min at 100°C. Aliquots of the NaOH digest were taken for protein determination according to Lowry et al. (1951) and for radioactivity which was done by Cerenkow counting in an Intertechnique SL-30 liquid scintillation counter. At least lo4 counts were recorded for each sample. Isolation of oocy te cortices and endoplasm Cortices were isolated according to the modification of Sakai’s procedure (1960) described by Kishimoto et al. (1976): oocytes were collected by centrifugation, washed rapidly with 0.1 M MgC12 and subjected to 8 strokes of a hand homogeniser

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fitted with a teflon pestle in ice-cold 0.1 M MgCla. A pellet was obtained by centriftguation at 3000 rpm for 1 min. The first supernatant was collected (‘endoplasmic fraction’) and the pellet washed once more with 0.1 M MgClz to obtain isolated cortices. For 32P incorporation measurements, cortices and endoplasm were processed in the same manner as intact oocytes. The KCl-soluble fraction was obtained from isolated cortices according to the procedure of Murofushi (1974) for sea urchin oocytes. ~otein case activities These were determined by measuring the initial rate of 32P transfer from [T-~~P] ATP to cold TCA-insoluble material according to a method described elsewhere (Guerrier et al., 1975). Protein phosphatase activities 32P-labelled oocytes were fixed in ice-cold 15% TCA I h after I-MA addition. Precipitated proteins were extensively dialyzed until pH 7 was reached against a cold solution (4°C) containing 50 mM phosphate buffer, pH 7.5,lO mM MgCla and 1.35 mM DTT. Labelled proteins were added to homogenates and incubated at 25V. Reactions were terminated by adding ice-cold 10% TCA. Radioactivity of the supernatant was measured after protein precipitation and centrifugation. Statistical significance of the results Except for the experiments from figs. 3 and 4, which were done only in duplicates, at least five separates were performed for any experiment presented here, yielding a precision more than adequate for our purposes. In no case did standard deviation exceed 6% of the mean value. For the sake of clarity, only mean values were indicated on the figures.

RESULTS Kinetic analysis of 32Pincolporation into proteins of intact oocytes after hormonal stimulation or mimetic treatment The results of a typical experiment are shown in fig. 1. Specific radioactivity of proteins increases very rapidly after addition of 5 X IO-’ M I-MA, and becomes si~i~cantly different from the controls 5 min after hormone addition (control oocytes: 1197 + 48 cpmlmg proteins; hormone-st~ulated oocytes: 1947 rt 70 cpm/mg proteins; n = 9). Specific radioactivity reaches a steady-state value after 30-45 min, at which all triggered oocytes have already undergone germinal vesicle breakdown, most of them between the 18th and the 20th minute after exposure (9 experiments). Similar results were obtained when 2 X lo-* M dithiothreitol (DTT), a compound which has been shown to mimic l-MA action in reinitiating meiosis

P. Cuerrier et al.

Fig. 1. Evoluiion of the 32P-specific radioa~fivity of the TCA-~so~uble protein fraction obtained from ~a~~~a~~~~~~s glaciaiis unsiiRlu~ted and stimulated oocytes. Oocytes were incubated in medium containing 2 @B/ml [32P]P~4~z~a for 70 min, washed carefully in calciumFree sea water and divided into 3 batches. One served as control (0) while the other two were triggered to reinitiate meiosis using respectively 5 X 10e7 M I-methyladenine (I-MA) (A) or 2 X 10m2 M dithiothreitol (DTT) (0). 1 ml aliquots were collected at the specified times and proeessed as indicated in Materials and Methods. 32P counts incorporated into proteins accounted for l-4% of the total radioactivity taken up by the oocytes.

of starfish oocytes (Kishimoto and Kanatani, 1973), was substituted for l-MA in this experiment. However, in this case GVBD occurred in most oocytes about 25 min after DTT addition. Correlationbetween meiosisreinitiationand stimulationof “2Pincorporationinto pr0tt?inS

Biological activity of I-MA depends on its concentration according to a sigmoida1 law with threshoid value at about 1-2 X 10-a M and maximal response reached with 1-2 X IO--’ M. High concentrations are not ~hibito~ (Do&e et al., 1976). The results in fig. 2 show that the increase of the 32P incorparation into proteins as well as the steady-state level of protein phospho~~ation fo~ow~ng hormonal stimulation are likely to depend on I-MA concentration according to a rather similar law. The small increase of specific activity recorded with 2 X IOq8 M I -MA might be accounted for by that small, very sensible part of oocyte population (about

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Fig. 2. Deper&enceof the ~iimu~a~on of profein p~o~~~#r~~~iju~ on the concentration of l-MA in &act oocytes. Chcytes preloaded for 100 min were carafuUy washed and subjected TVvarious I-MA concen~ations: * control oocytes; o 2 X IOU8 M I-MA; k 2 X lo-’ M L-MA; 92x10 -f~ M l-MA; + 2 X tWs M I-MA.

We have shown that the presence of 1-MA is required ior only 4 min 30 s at 24’C whiie germinal vesicle breakdown occurs 18 rnin afzer the beginning of 1-MA treatment {Guerrier and DC&, 1975). The dependence of protein phos~hory~atjon on the continuous supply of 1-MA was therefore studied by eiiminating the hormone at various time intervals after its application, It is obvious from fig: 3 that the presence of 1-MA is not required during the entire time of nuclear n~aturation fvr eE6cient protein phos~~o~~at~on COoccur since the level attainer was almost the same when oocytes were exposed 7 min, 11 min, 18 min or continuously to I-MA. Nevertheless the level of’ protein phosphorylation attained was sharply decreased

P. Guerrier et al.

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Fig. 3. Influence of the time of contact with l-MA on both protein phosphorylation and biofogicaI response of Marfhasterimglacialisintact oocytes. Oocytes preloaded for 90 min and washed carefully were incubated either in the absence (0) or in the presence (0) of 5 X lo-’ M l-MA. 10 ml aliquots were taken respectively at 4 (X), 7 (A), 11 (0) or 18 (v) min after l-MA addition. In order to remove the hormone, these aliquots were immediately centrifuged (7 s), the pellets washed rapidly with 10 ml calcium-free sea water and resuspended in 10 ml of the same medium. At the indicated times, 1 ml aliquots of each batch of oocytes were injected into 15% cold TCA and processed as indicated in Materials and Methods. Meiosis reinitiation was triggered at 13% after 4 min of contact, at 95% after 7 min and at 100% after 11 min of contact with l-MA.

when l-MA was eliminated after 4 min, that is to say after a time period which allows only a few oocytes (13%) to undergo meiosis (T = 20°C). Although the sequence of events between I-MA binding to the membrane receptors and the breakdown of the germinal vesicle does not include compulsory synthesis of specific proteins, we have shown elsewhere (Cuerrier and DorBe, 1975) that relatively high concentrations of emetine, cycloheximide and puromycin were able to inhibit GVBD, and that these inhibitions were neutralized by increasing l-MA concentration. Competitive inhibitions of the biological response were also obtained with various methylxanthines, including caffein (DorCe et al., 1976). Fig. 4A and 4B show that inhibition of GVBD is always associated with a noticeable decrease of the rate of 32P incorporation into proteins with regard to stimulated

Fig. 4. Effects of various inhibitors on meiosis reinitiation and protein phosphorylation, using 32P preloaded oocytes. Two different coneentrations of I-MA were used: 2 X 10M7 M and 5 X 1C5 M. CVBD indicates that the treatment resulted in more than 95% meiosis ~einit~tion. In each graph, the symbol s refers to control unstimulated ooeytes and o to 2 X 10v7 M i-MA stimulated oocytes. A. Effects of emetine: + 2 X 10e7 M I-MA and 100 &ml cmetine, a concentration which abolishes protein synthesis:r 2 X 10m7 M I-MA and 400 &g/ml emetine; A 5 X 10m5 M I-MA and 400 #g/ml emetine. Arrow points to the simultaneous addition of emetine and I-MA. 3.2 X 10m3 M caffein was added simuttaneousfy (arxow) with 2 X 20v7 M l-MA (A) or 5 X 10v5 M l-MA (A). G. In this experiment, 1 mg/ml leupeptin was added to the oocytes 1 h before the addition (arrow) of l-MA, either-2 X 1OF7 M {A) or_5 X IO-’ M (A). D. 2 X 10v3 M diamide was added (1st arrow) 5 min befoxe the addition (2nd arrow)

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oocytes. Further, it has been shown by Clark and Kanatani (1975) that two protease inhibjtors of microbial origin, ~eupeptin and antipain, abolish meiosis reinitiation which is normally induced by 2 X lo-’ M 1-MA, and we have found that this inhibition can be reversed by slightly increasing the hormone concentration. in addition, fig. 4C shows that in this case also, suppression of the biological response is linked with a drastic reduction of 32P incorporation into proteins with regard to oocytes which will usually undergo meiosis reinitiation. Finally, fig. 4D shows that diamide, a potent oxidizing agent for thiol groups and which is antagonized by DTT (von Tersch et al., 1975; Pillion et al., 19’76), suppresses both stimulation of 32P incorporation into proteins and the biological response when I-MA is used at low concentration. In this case also, increasing I-MA concentration releases oocytes from diamide inhibition with regard to both biochemical and biological responses.

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Fig. 5. Time course of 32P incorporation into proteins of both cortical and endoplasmic fractions prepared from preloadcd control or stimulated oocytes. l-MA 10m6 M was added (arrow) to the oocytes 20 min after resuspension into 32P-free sea water. Samples were processed as indicated in Materials and Methods. l cndoplasm from control; o cndoplasm from stimulated oocytes; A cortex from control; A cortex from stimulated oocytes.

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Differential pattern of protein phosphotylation in cortex and in endoplasm Since appearance of MPF has been assumed to be a consequence of changes occurring in the cortical region after 1-MA interaction, we investigated the possibility that the time course of 32P incorporation into proteins could be different in the cortical and in the endoplasmic region of I-MA st~tllulated oocytes. Fig. 5 shows that stimulation of 32P incorporation into proteins occurs in both the cortex and the endoplasm. However, specific activity of proteins increases much more rapidly in the cortex. Steady-state occurs after about 0.5 h in endoplasm whereas it is already reached between the 8th and the 10th minute in cortex. It was further found, in this experiment, which was performed at 2O”C, that oocytes collected 10 min after starting the hormonal treatment and resuspended in hormone-free medium were already triggered to undergo meiosis whereas they failed to do so when collected after only 7 min. Since these later experiments supported the view that l-MA might enhance preferentially the phosphorylation of cortical proteins, perhaps accessible from outside to ATP, 10e5 M [T-~*P] ATP was supplied externally to intact oocytes and 32P incorporations into proteins of control and l-MA stimulated oocytes were compared. No difference in protein iabelling was found between control and stimulated oocytes, although 32P incorporation into proteins was readily measurable (IO3 cpm 32P incorporated in less than 15 minjmg protein).

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Fig. 6. Time course of protein phosphoryiation in homogenates prepared before (a) or 5 min after supplying 1W6 M l-MA to intact oocytes f*). A refers to endogenous pho~horylation, B to histone phosphorylation, which was estimated as the difference between the overall protein phosphorylation with histone supplemented homogenates and endogenous phosphorylation.

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Stimulation of protein kinase activity after hormonal treatment Since the above results suggested that l-MA might act by stimulating protein kinase activity, we measured the ability of [T-~*P] ATP supplemented homogenates to support both phosphorylation of their own proteins (endogenous phosphorylation) and phosphorylation of exogenous substrates (casein or histones). The results of a typical experiment are shown in fig. 6. Initial rate of endogenous phosphorylation as well as plateau level were already increased 20%50% 5 min after hormone addition to intact oocytes (18 experiments). Moreover stimulation was still higher with histone or casein supplemented homogenates. Since this might be due either to (1) an increase of protein kinase activity or (2) a decrease of protein phosphatase activity, 32P-labelled proteins were prepared from TCA-fixed labelled oocytes and the rates of 32P release from this substrate compared after its addition to control and stimulated homogenates. No difference in protein phosphatase activity was observed between the two types of homogenates. The extent of protein kinase stimulation was then studied as a function of time elapsed after l-MA addition to intact oocytes. Fig. 7 shows that homogenates did not differ from control when prepared 2 min after l-MA addition. However, protein kinase increased sharply between the 2nd and the 5th minute. Maximal rates of histone (6-fold that of control) and casein phosphorylation (3-fold that of control) were reached 5 min after hormone addition. Phosphorylation rates remained much higher than controls up to the extrusion of the first polar body. No modifications occurred at GVBD. It is clear from fig. 7 that a lesser stimulation of phos-

Fig. 7. Time course of l-MA effect on histone kinase activity (o), casein kinase activity (+) and endogenous protein kinase activity (0) using whole oocyte homogenates prepared after various time periods of incubation with 10h6 M l-MA, which was added at zero time. Germinal vesicle breakdown (GVBD) and first polar body cvtrusion (1st p.b.) are indicated by arrows.

Protein phosphorylation

during starfish meiosis

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Fig. 8. Time course of endogenous protein phosphorylation in endoplasmic fractron (A), cortical fraction (B) and KCI-soluble cortical fraction (C) prepared before (0) or 10 min after supplying lo@ M l-MA to intact oocytes (A). The left-hand scale refers to A only, the right-hand one to B and C.

phorylation occurred with endogenous substrates as compared to exogenous substrates, especially histones. Since the in vivo experiments suggested that the pattern of protein phosphorylation might be different in oocyte cortex and endoplasm, both fractions were isolated 10 min after l-MA addition to intact oocytes and checked for endogenous protein phosphorylation. The result shown in fig. 8B and 8C suggests that cortical proteins from stimulated oocytes were phosphorylated in vitro to a lesser extent than those from control oocytes (11 experiments). On the other hand, fig. 8A shows that hormonal treatment enhanced the in vitro phosphorylation of endoplasmic proteins, confirming the results already obtained using whole oocyte

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Fig. 9. Time course of l-MA effect on histone (0) in cortical fractions prepared after various oocytes.

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homogenates. Similar results were obtained from oocytes which were kept for longer periods of time in the presence of l-MA (fig. 9). Nevertheless we have found that even in cortex, protein kinase activity towards exogenous substrate (histone) was enhanced after hormone treatment.

DISCUSSION Our results show that l-MA (or DTT) application increases the specific activity of 32P-labelled proteins in starfish oocytes externally supplied with [32P] P04H2Na. This finding cannot be explained on the basis of (1) the increase of specific activity of intracellular ATP, since the 32P was eliminated from the medium before hormone stimulation; and furthermore, variations in permeability do not precede GVBD but occur simultaneously with it (DorCe and Guerrier, unpublished results): (2) the appearance of newly synthesized protein substrate, since suppression of protein synthesis does not inhibit meiosis reinitiation nor the increase of protein specific activity; or (3) a difference in protein phosphatase activity, since there is none between controls and stimulated oocytes, so it does appear that our results are due to a true stimulation of protein phosphorylation. This interpretation is supported by our finding that protein kinase activity is much higher in homogenates prepared from hormone-stimulated oocytes than from control oocytes. The possibility that the increase of protein phosphorylation might be due partially to some structural changes of protein substrates rather than protein kinase (Bylund and Krebs, 1975) cannot, however, be entirely ruled out. That stimulation of protein phosphorylation is a prerequisite for meiosis induction is strongly indicated by our results which show stimulation of protein phosphorylation and frequency of meiosis reinitiation to be almost identical in oocyte preparations with respect to concentration dependence, kinetics, reversibility and sensibility to various inhibitors. That stimulation of protein phosphorylation is a very early consequence of l-MA interaction with its membrane receptors is indicated by our results which show that this stimulation is already noticeable 2 min after addition of the hormone and is already irreversibly triggered at the end of the ‘hormone-dependent phase’ (Cuerrier and Dome, 1975). The finding that any inhibitor of the biological response decreases the stimulation of protein phosphorylation is highly consistent with the interpretation of our results since oocytes are released from any inhibition by raising hormone concentration. This seems to suggest that all inhibitors tested probably act either by interfering with 1-MA binding to its receptors or by inhibiting some early event which results from this binding. It appears that stimulation of phosphorylation of cortical proteins may be of major importance. We have found that protein kinase activity and the rate of stimulation of protein phosphorylation is much higher in the cortex than in the endoplasm. The consequence is that protein phosphorylation reaches its steady-state

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value much earlier in the cortex than in the endoplasm. The reduced rate of endogenous phosphorylation observed following hormone stimulation in the cortex (or in a KC1soluble fraction extracted from it) but not in endoplasm is readily accounted for by the lack of substrate available for phosphorylation in the cortices. On the other hand, other experiments support the view that protein phosphorylation in the endoplasm might be a consequence of the changes taking place in the cortex because the concentration of endoplasmic phosphorylated proteins will stop increasing if 1 -MA is eliminated before maximal cortical phosphorylation, whereas it continues to increase normally if the hormone is eliminated later on. The nature of the target for protein phosphorylation in the cortex remains unknown. The vitelline membrane, a potential substrate for phosphorylation, does not appear to play any signi~cant role in meiosis reinitiation. Our experiments with vitelline membrane-free oocytes indicate that these oocytes do not show any difference from normal oocytes with respect to biological responses and stimulation of protein phosphorylation on exposure to l-MA (DorCe and Guerrier, unpublished results; Moreau and Cheval, 1976). When [i-“‘PI ATP was added directly to oocytes, 32P incorporation into proteins could be measured easily, but no difference in protein labelling between control and hormone-stimulated oocytes was observed. It appears likely that neither vitelline membrane proteins nor those plasma membrane proteins which are accessible for phosphorylation by external ATP are targets for l-MA stimulated protein phosphorylation. It should be noted that the plasma membrane does not normally elicit translocation of intact nucleotides (Pourquie and Heslot, 1971). We have recently shown that the injection of phosphorylase kinase (an enzyme that catalyses the phosphorylation of both contractile proteins and phosphorylase b), but not injection of phosphorylase a, readily induces meiosis reinitiation in ~e~~~~~ taevis oocytes (Moreau et al., 1976). Meiosis reinitiation has been shown to be linked with microvilli retraction (Truj~lo~enoz and Sotelo, 1959; Odor, 1960; Stegner and Wartenberg, 1961; Wartenberg and Schmidt, 1961; Gwyun and Jones, 1971; Hirai et al., 1971). It would be of interest to obtain information about phosphorylation of cortical contractile proteins in the hope of learning if they are in any way related to the system we are studying. On the other hand, Kishimoto et al. reported recently (1976) that the sulfhydryl content of oocyte-cortex protein had increased about 9% 10 min after 10e5 M 1-MA addition. In order to elucidate the relationships between 1-MA interaction with its membrane receptors and its two early biochemical consequences occurring in the vicinity of the plasma membrane, we can now ask the question: is reduction of protein disulfide bonds related to stimulation‘ of protein kinase activity? Although the possibility cannot be ruled out that enzymes like glutathione reductase which catalyze the reduction of protein disulfide bonds might be regulated by phosphorylation and dephospho~lation, it seems more likely to assume that activation of cortical protein kinase involves reduction of protein disultide bonds. This would explain why disultide-reducing agents such as DTT induce both meiosis reinitiation and its prerequisite, the stimulation of protein phosphorylation, where-

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as potent oxidizing agents reacting with thiol groups, like diamide, abolish both biological and biochemical response to 1-MA. Progress in analyzing the mechanism of hormonal control of meiosis reinitiation in starfish oocytes will provide a very suitable biological system to understand how enzyme-catalyzed chemical modifications are involved for the amplification of regulatory signals.

ACKNOWLEDGEMENTS We are very grateful to Mrs. C. Guerrier for excellent technical assistance and for drawing the figures and to Dr. E.C. Preddie, from McGill University in Montreal, for reading the manuscript. We greatly acknowledge the financial support from the C.N.R.S. (ATP 1890) and the D.G.R.S.T. (ACC 659-1340).

REFERENCES Bylund, D.B. and Krebs, E.G. (1975) J. Biol. Chem. 250,6355-6361. Clark, T.G. and Kanatani, H. (1975) Biol. Bull. 149,423. Doree, M. and Guerrier, P. (1975) Exp. Cell Res. 96,296-300. Do&e, M., Guerrier, P. and Leonard, N.J. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1669-1673. Guerrier, P. and Doree, M. (1975) Dev. Biol. 47,341-348. Guerrier, P., Doree, M. and Freyssinet, G. (1975) C.R. Acad. Sci. (Paris) 281,1475-1478. Gwyun, I. and Jones, P.C.T. (1971) Z. Zellforsch. Mikrosk. Anat. 113,388-395. Hirai, S., Kubota, J. and Kanatani, H. (1971) Exp. Cell Res. 68, 137-143. Kanatani, H. (1973) Int. Rev. Cytol. 35,253-298. Kanatani, H. and Hiramoto, H. (1970) Exp. Cell Res. 61,280-284. Kanatani, H. and Shirai, H. (1971) Dev. Growth Differ. 13,53-63. Kishimoto, T. and Kanatani, H. (1973) Exp. Cell Res. 82,296-302. Kishimoto, T. and Kanatani, H. (1976) Nature (London) 260,321-322. Kishimoto, T., Cayer, M.L. and Kanatani, H. (1976) Exp. CeB Res. 101, 104-110. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol.Chem. 193,265275. Moreau, M. and Cheval, J. (1976) J. Physiol. (Paris) 72,293-300. Moreau, M., Guerrier, P. and Doree, M. (1976) J. Exp. Zool. 197,435-442. Murofushi, H. (1974) Biochim. Biophys. Acta 354,260-271. Odor, D.L. (1960) J. Biophys. Biochem. Cytol. 7,567-574. Pillion, D.J., Leibach, F.H., von Tersch, F. and Mendicino, J. (1976) Biochim. Biophys. Acta, 419,104-111. Pourquie, J. and Heslot, H. (1971) Genet. Res. 18,29-44. Sakai, H. (1960) J. Biophys. Biochem. Cytol. 8,603-615. Schmidt, G. and Thannhauser, S.J. (1945) J. Biol. Chem. 161,83-89. Stegner, H.E. and Wartenberg, H. (1961) Z. Zellforsch, Mikrosk. Anat. 53, 702-713. Von Tersch, F.J., Mendicino, J. Pillion, D.J. and Leibach, F.H. (1975) Biochem. Biophys. Res. Commun. 64,433-440. TrujilloCenoz, 0. and Sotelo, J.R. (1959) J. Biophys. Biochem. Cytol. 5,347-350. Wartenberg, H. and Schmidt. W. (1961) Z. Zellforsch. Mikr. Anat. 54, 118-146.