Lithium inhibits amplification or action of the maturation-promoting factor (MPF) in meiotic maturation of starfish oocytes

Experimental Cell Research 147 (1983) 41-50

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Lithium Inhibits Amplification or Action of the Maturation-Promoting Factor (MPF) in Meiotic Maturation of Starfish Oocytes ANDRE? PICARD and MARCEL

DORfiE

CRBM-CNRS, INSERM U249, Facultt de Mkdecine, B.P. 5015, F-34033 Montpellier Cedex, and Station Biologique, F-29211 Roscoff, France

SUMMARY Microinjection of LiCl reversibly inhibits hormone-induced meiotic maturation of starfish oocytes. Microinjection of NaCl (even in ouabain-treated oocytes) or KCI, or external application of LiCl have no such effect. Blockade of meiotic maturation by Li+ occurs even when microinjection is performed after the hormone dependent period has ended, that is the period during which the hormone must be present in the medium in order that meiosis can take place. Li+ microinjection prevents oocytes from meiosis reinitiation following transfer of cytoplasm taken from maturing oocytes, which contain a maturationpromoting factor (MPF). Cytoplasm taken from Li+-injected and hormone-treated oocytes does not trigger meiosis reinitiation when transferred in control immature oocytes. Intracellular pH does not change following LiCl microinjection. Simultaneous microinjection of either K+, Na+, or EGTA does not prevent Li+-dependent inhibition in oocytes.

In starfish, oocytes are arrested at the prophase stage of meiosis. Meiosis is reinitiated by a hormone originating from the follicle cells [ 11, which has been identified as 1-methyladenine (1-MeAde) [2]. The hormone acts on stereospecific receptors localized on the plasma membrane [3-51 to produce an amplifiable cytoplasmic maturation-promoting factor (MPF), which releases the oocytes from the prophase block, and to induce complete meiotic maturation [6]. In the course of our investigations about the mechanism of meiotic maturation, we found that starfish oocytes microinjected with lithium ions failed to reinitiate meiosis in response to l-MeAde addition. This result was of interest, since it is well-known that lithium also induces specific disorders (‘vegetalization’) in morphogenetic gradients of echinoderm embryos [7-91, and since the local application of l-MeAde has been shown recently to modify the polarity of starfish oocytes [lo]. The aim of this work was to describe the effects of lithium microinjection on hormone-induced meiotic maturation in starfish oocytes. The results suggest that lithium might inhibit meiosis reinitiation by interfering either with MPF amplification or action.

MATERIAL

AND METHODS

Chemicals and artificial sea waters I-MeAde, ethylene glycol bis (#I-aminoethylether)-N,iV’-tetraacetic acid (EGTA), piperazine-N,N’his (t-ethane sulfonic acid) (PIPES), choline chloride, were purchased from Sigma, USA, LiCl and Tris (hydroxymethyl)~aminomethane were obtained from Merck, Germany. In some experiments, we used artificial sea waters. ‘Normal’ artificial sea water had the following composition: 451 mM NaCl, 9.92 mM KCl, 11.27 mM MgCla, 7.81 mM MgS04, 10 mM CaCl*, 2 mM Tris-HCI pH 8.15. Na-free artificial sea water was prepared by substituting either LiCl, choline chloride or Tris-HCl for NaCl.

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Picard and Dorte

Preparation

Exa Cell Res 147 (1983)

of oocytes and microinjections

Fully-grown prophase-blocked oocytes were prepared free of follicle cells by washing them several times in Ca*+-free artificial sea water, and finally transferring them into Millipore-filtered natural sea water. Microinjections were performed according to the method of Hiramoto [l I]. To prevent leakage of salines, the tip of the micropipet was sealed with a small amount of silicon oil after introduction from the tip of the material to be injected.

Extraction

of MPF

MPF-containing extracts were prepared essentially according to Wu & Gerhart [42], except that the standardized solution for the extraction of MPF contained 144 mM sodium /I-glycerophosphate, 36 mM potassium EGTA, 27 mM MgC12, 1.8 mM DTT, 200 mM sucrose and 250 mM KC1 at a final pH of 7.3.

Measurements

of intracellular

pH (pHi)

Intracellular pH was monitored with a neutral carrier-based hydrogen ion-selective microelectrode. The carrier was a generous gift of Professor Simon, from the Swiss Federal Institute of Technology, Zurich. The H+-specific microelectrodes were prepared according to Amman [40]. Conventional microelectrodes were filled with 3 M KC1 and broken to resistances of 10-20 MB. EMF measurements were made with a FET operational amplifier (OPA 104 AM, Burr Brown, USA; input impedance 10” Q/O.8 pF differential; 10”/1.6 pF common mode; input bias current ~300 fA). The amplifiers were mounted on top of electrodes. The electrodes assemblies were located inside a Faraday cage. The difference signal between the membrane potential electrode and an extracellular chlorinated silver wire was displayed on a channel of a slow speed chart recorder. The difference signal between the H+-selective microelectrode and the conventional membrane potential electrode was displayed on a second channel.

Table 1. Effect of KCI, NaCl subsequent addition of I-MeAde

or LiCl

microinjection

on GVBD

induced

by

No. of oocytes with GVBD/no. of injected oocytes Injected chloride”

Delay between injection and hormone addition

None (control) K+

I-MeAde cont. (M)

0.5 h after 1-MeAde addition

4 h after I-MeAde addition

316 616

316 616

o/4 316 515

o/4 316 515

214 515

214 515

5x10-7 1o-6 1o-5 10-4

o/4 o/5 o/5

o/4 315 315

3/10

10110

5x10-7 10-6 1o-4

015 O/6 O/6

o/5 O/6 616

10-7 2x 10-7

< 10 min -

10-7 2x10-7

5x lo-’ Nat


Li+


Li+

4h -

2x

10-7

5x lo-’

a 50 pl of 0.8 M chloride- 10 mM PIPES-KOH,

pH 6.8.

Exp Cell Res

147 (1983)

Lithium and meiotic maturation

Fig. 1. Effect of the concentration of the microinjected LiCl solution on meiosis reinitiation induced by various I-MeAde concentrations. Percentages of germinal vesicle breakdown estimated 30 min after hormone addition to oocytes microinjected (less than 10 min before hormone addition) with 50 pl of either 0.2 M LiCl, 0.5 M LiCl, or 0.8 M LiCl solutions adjusted at pH 6.8 with 10 mM PIPES-KOH. ‘Control’ refers to uninjected oocytes.

RESULTS Effect of injecting lithium on hormone-induced meiosis reinitiation When fully-grown immature oocytes (internal volume about 2 nl), microinjected either with 50 pl of 0.8 M NaCl- 10 mM PIPES-KOH, pH 6.8 or 50 pl of 0.8 M KCl-10 mM PIPES-KOH, pH 6.8, were transferred, simultaneously with control uninjected oocytes, in natural sea water containing 1-MeAde at various concentrations, their sensitivity to the hormone was only very slightly decreased with respect to the control oocytes. 100% of the KCl- or NaCl-injected oocytes reinitiated meiosis when the I-MeAde concentration was 5~ 10m7M (table 1). In all cases germinal vesicle breakdown (GVBD) occurred less than 20 min after hormone addition. In contrast, when oocytes received 50 pl of 0.8 M LiCl (adjusted at pH 6.8 with 10 mM PIPES-KOH) and were subsequently transferred in natural sea water containing various concentrations of hormone (from 10m7to 10m5M) GVBD did not occur or was strongly delayed. When the hormone concentration was raised to 10m4M, GVBD was still delayed but occurred in all oocytes. Whereas the oocytes slowly escaped from the blockade induced by lithium microinjection when kept in the presence of a high hormone concentration, the inhibition was not reversed and was even increased when the microinjected oocytes were first left as long as 4 h in natural sea water before hormone addition (table 1). As shown in fig. 1, inhibition of hormone-induced meiosis reinitiation was highly dependent on the lithium concentration. In fact, strong inhibition was observed only when the intracellular Li+ concentration was higher than 12.5 mM, assuming a homogeneous distribution of the microinjected material. It has been shown that the hormone only has to be present in the medium for a few minutes (‘the hormone-dependent period’) for meiosis to occur [12, 131. Therefore we investigated next whether Li+ still inhibits meiosis reinitiation when microinjection takes place after termination of the hormone-dependent period. Oocytes were taken at various times after addition of 2x 10e7 M l-

43

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Picard and Dot-&e

Exp Cell Res 147 (1983) GVBD 1

lOO.%

SO-

07 0

.

* 5 Time after

IO I-MeAdo

addition

Fig. 2. Effect of either intracellular microinjection of LiCl or of elimination of the hormone, both at various times following addition of 2x lo-’ M I-MeAde, on meiosis reinitiation. I-MeAde was added at zero time. At the indicated times, oocytes were either transferred in sea water without lMeAde (A) or microinjected with 50 pl of 1 M LiCl adjusted at pH 6.8 with 10 mM PIPES-KOH (0). Percentages of GVBD were estimated 0.5 h after hormone addition .

I5 (min)

MeAde and either kept in continuous presence of the hormone but injected with 50 pl of 1 M LiCl- 10 mM PIPES-KOH, pH 6.8, or transferred without injection into hormone-free natural sea water. 100% of the uninjected oocytes underwent GVBD 13 min after hormone addition when kept in the presence of l-MeAde for at least 5 min. In contrast, the nuclear envelop had not disappeared 30 min after hormone addition, even when LiCl was injected as late as 12 min following hormone addition, thus only 1 min before GVBD occurred in control oocytes (fig. 2). Oocytes microinjected less than 10 min after hormone addition remained definitively arrested (unless the hormone concentration was raised), but oocytes receiving Li+ 11 and 12 min after hormone addition underwent GVBD about 2 h later. EfSect of extracellular addition of lithium on hormone-induced meiosis reinitiation Oocytes were transferred either in normal artificial sea water or in sodium-free artificial sea water, where either lithium, choline or Tris were quantitatively substituted for Naf. The sensitivity of the oocytes to 1-MeAde was estimated in each case. It was found that elimination of Na+ from sea water somewhat increased the sensitivity of starfish oocytes to I-MeAde. The concentrations of hormone required for 50% of the oocytes to undergo GVBD were found to be 4x 10m8M and 9x 10e8 M in sodium-free artificial sea water and normal artificial sea water respectively. No significant difference was found whether sodium was replaced by lithium, choline, or Tris, even when the oocytes were transferred in various Naf-free artificial sea waters 0.5 h before hormone addition (data not shown). Effect of injecting lithium on MPF-induced meiotic maturation It has been shown that 1-MeAde acts on the cell surface to produce a maturationpromoting factor (MPF), whose presence in cytoplasm has been demonstrated only after the termination of the hormone-dependent period, and which induces GVBD when injected into fully-grown immature starfish oocytes [6]. Therefore, the effects of the intracellular microinjection of lithium on oocyte maturation induced by MPF were next examined. 20-40 min after hormone addition to

Exp Cell Res

Lithium and meiotic maturation

147 (1983)

control oocytes, 200 pl of cytoplasm was taken from maturing oocytes and injected into immature oocytes which had been microinjected previously either with 50 pl of 1 M LiCl or with 50 pl of 10 mM PIPES-KOH, pH 6.8 (the vehicle of LiCl). GVBD occurred without exception less than 15 min after cytoplasm transfer in the 10 oocytes microinjected with the buffer alone. In contrast, only one out of the 10 LiCl-microinjected oocytes had undergone GVBD 1 h after cytoplasm transfer. However, two additional oocytes escaped later from the Li+dependent blockade (table 2). In another experiment, eight oocytes were microinjected with 50 pl of 0.8 M LiCl- 10 mM PIPES-KOH, pH 6.8 from 1 to 8 min after external addition of 4~ 10e7 M l-MeAde. 3U7 min later 200 pl of cytoplasm was taken from these Li+-arrested oocytes and transferred (in the absence of hormone) in control immature oocytes. About 1 h after cytoplasm transfer GVBD occurred in one of eight recipient oocytes, whereas the other recipient oocytes remained arrested. Nevertheless they underwent GVBD when transferred into sea water containing 10m4M l-MeAde. In this experiment no correlation was observed between the time of LiCl injection and the ability of the cytoplasm to bring about GVBD in recipient oocytes. Possible mechanisms of lithium-induced inhibition Alkalinizing intracellular pH (pHi) inhibits 1-MeAde-induced meiosis reinitiation in starfish oocytes [14, 15,411. On the other hand, Na+/H+ exchangeshave been shown to be involved in the regulation of pHi in many cells, including echinoderm eggs [16, 171.Since lithium was perhaps competing with Na+ and interfering with Na+/H+ exchange, we investigated the possible effect of the microinjection of 50 pl of 1 M LiCl- 10 mM PIPES-KOH, pH 6.8 on pHi. As shown on fig. 3, pHi did only change slightly from its resting value of 6.92 to 6.86, due to the PIPES buffer. Lithium by itself had no effect on pHi. Control experiments showed that 20 mM Li+ does not interfere with pH measurementsby the neutral carrier-based hydrogen ion-selective microelectrode (data not shown). Since Li+ has been shown to compete with intracellular cations in several systems [l&20], we tested whether simultaneously microinjecting K+, Na+, or Mg2+ might counteract Li+ in inhibiting hormone-induced meiosis reinitiation. Table 2. Effect of injecting lithium on MPF-induced meiotic maturation GVBDb Material injected in MPF-recipient oocytes” 10 mM PIPES-KOH, pH 6.8 1 M LiCl- 10 mM PIPES-KOH,

pH 6.8

1 h after MPF transfer

4 h after MPF transfer

lO/lO l/10

lO/lO 3/10

’ MPF-injected oocytes were microinjected with LiCl or its vehicle (injected volume, 50 pl) 14 min before transfer of 200 pl of MPF-containing cytoplasm taken 20-40 min after hormone addition from hormone-treated oocytes. b No. of oocytes with GVBD no. of injected oocytes.

45

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Picard and Do&e

Exp Cell

-fl T f

mv pH 0 8.15

20..

so.. .?.I5 60’:1.0 -6.9 70”

-6.6

!

Fig. 3. Original records of pHi (upper truce) and membrane potential (lower) in an immature oocyte microinjected with 50 pl of 0.8 M LiCl-10 mM PIPES-KOH, pH 6.8. After calibrating the pH microelectrode, both the conventional and the pH microelectrodes were introduced in natural sea water buffered at pH 8.15 with 10 mM HEPES-NaOH. The conventional microelectrode was inserted first (arrow I); therefore the pH trace was deflected downwards because the pH record is the difference signal between the H+-sensitive and the conventional microelectrodes. The pa-sensitive electrode was inserted next (arrow 2), then the LiCl-containing micropipet (arrow 3). Microinjection was performed after stabilization of the pH signal (it& Finally the pH microelectrode (arrow 4) and the conventional electrode (arrow 5) were removed from the oocyte.

Although these experiments did not definitively rule out the possibility that Lif was competing with any of these cations, since the affinity of the Li+sensitive component for Li+ may be too great for an effect to be observed, we found that the inhibition by Lif was not decreased when oocytes received 1 M LiCl simultaneously with either 1 M KCl, 1 M MgC12 or 1 M NaCl (data not shown). Our result that elimination of Na+ from sea water had a facilitating effect on hormone-induced meiosis reinitiation supported the alternative hypothesis that a high level of intracellular Na+ was perhaps detrimental for meiosis reinitiation. Therefore we considered the possibility that microinjected Li+ might mimic and amplify such a presumed inhibitory effect. This hypothesis was not completely ruled out by our previous result that microinjection of Naf only slightly decreased oocyte sensitivity to 1-MeAde, since failure of microinjected Na+ to

Res 147 (1983)

Lithium and meiotic maturation

Exp Cell Res 147 (1983)

block meiosis reinitiation was perhaps due to its rapid elimination from the oocyte by the hormone-stimulated Na+ pump [5, 221. Therefore oocytes were first kept in natural sea water containing 2 mM ouabain, then 5x10-' M lMeAde was added and the oocytes microinjected l-10 min later with 50 pl of 800 mM NaCl- 10 mM PIPES-KOH, pH 6.8. It was found that all the microinjected oocytes underwent GVBD 13-15 min after hormone addition, simultaneously with control uninjected oocytes, treated or not with ouabain (data not shown). Free Ca*+ has been shown to destroy the MPF activity of cytoplasmic extracts [21]. Although our results suggested that Lif might inhibit MPF amplification or action, we considered the possibility that Li+ might act in ouo in an indirect way, by increasing first the intracellular concentration of free Ca*+. Therefore we investigated the effects of injecting a Ca*+ -chelator, EGTA, simultaneously with LiCl. As shown in table 3, EGTA was not found to prevent oocytes from Li+induced inhibition of meiosis reinitiation. We also considered that Li+ could be acting to destroy the MPF activity directly. To investigate this possibility, a crude extract having MPF activity was prepared from maturing oocytes 30 min after 1-MeAde addition. An identical and small volume of either 1 M KC1 or 1 M LiCl was added to this extract. Twenty minutes later fully-grown immature oocytes (internal volume about 2 nl) were injected either with the Li+-free extract or with the extract made up to 25 mEq/l with respect to Li+. In both cases 100% of the recipient oocytes (lo/lo) underwent GVBD when 260 pl of extract was injected. When the microinjected volume was reduced to 160 pl, 40% of the recipient oocytes (4/10) underwent GVBD in both cases. DISCUSSION In the present work, we have shown that microinjection of Li+ in immature starfish oocytes at a final intracellular concentration higher than 12.5 mM prevents them from undergoing meiosis reinitiation following either hormone addition or MPF transfer. Such an effect was not observed when the same amount of Na+ or K+ was injected. Oocytes escaped slowly from the Lif-dependent

Table 3. Effect of the simultaneous microinjection inhibition of meiosis reinitiation Microinjected material (50 PO

800 mM LiCl in 10 mM PIPES-KOH, pH 6.8 800 mM LiCl- 100 mM EGTA in 10 mM PIPES-KOH, pH 6.8 10 mM PIPES-KOH, pH 6.8 100 mM EGTA in 10 mM PIPES-KOH, pH 6.8

of EGX4 on LP-induced

Delay from I-MeAde addition (5 x lo-’ M) to microinjection

GVBD”

l-10 min l-10 min

O/8 O/8

l-10 min l-10 min

717 J/J

0 No. of oocytes with GVBD no. of microinjected oocytes (GVBD occurred from 13 to 15 min after hormone addition in control oocytes). 4-838334

47

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Picard and Dorte

Exp Cell Res 147 (1983)

blockade, presumably due to leakage of Li+ from the oocytes, in the presence (but not in the absence) of I-MeAde. Since the hormone has been shown to stimulate the activity of the Na’ pump in starfish oocytes [S, 221, we believe that the Na+ pump is involved in pumping out Li+ from the oocytes. In agreement with this view, it has been shown that Li+ stimulates the inside Naf sites of the ATPase system in mammals [23]. It has been reported that Li+ might alter membrane permeability to Naf [24]. Although the rate of the unidirectional Nat influx has been shown to increase in Marthasterias glacialis oocytes following 1-MeAde addition [25], our observations that elimination of Naf from sea water does not inhibit, but rather facilitates the action of I-MeAde, demonstrate that a net influx of Na+ is not required to trigger meiosis reinitiation. On the other hand, Naf microinjection did not release starfish oocytes from Lif-induced blockade of meiosis reinitiation. It is therefore doubtful whether Li+ might inhibit meiotic maturation by blocking Na+ channels, although such a membrane effect of Lif has been shown to occur in other oocytes [24]. Finally, Li+ cannot block meiosis reinitiation as a consequence of some possible inhibition of the Na+ pump activity, since starfish oocytes readily undergo meiosis reinitiation in the presence of ouabain, which abolishes Na+ pump activity [22]. Li+ has been shown to mimic Na+ in inducing efflux of intramitochondrial Cazi 126, 271. Although microinjection of Ca” readily inhibits hormone-induced meiosis reinitiation [28], such inhibition was, however, not observed when the intracellular concentration of Na+ was increased by at least 20 mM following microinjection of NaCl in ouabain-treated oocytes. Moreover, the Li+-dependent inhibition was not decreased when LiCl was injected simultaneously with the Ca*+-chelator EGTA. It is therefore doubtful whether a release of Ca2+ from mitochondria due to Li+ (or Na+) microinjection might account for the inhibition of hormone-induced meiosis reinitiation. We also ruled out the possibility that Li+ might act by changing pHi due to its possible interference with Nai/Hf exchange. It seems unlikely that Li+ might -inhibit interaction of 1-MeAde with its stereospecific receptors, localized on the plasma membrane and accessible only from the outside to the hormone, since external addition of Lif does not inhibit hormone-induced meiotic maturation. In addition, microinjection of Lif does not prevent starfish oocytes from demonstrating a distinct but specific response to the application of the hormone, the elevation of a fertilization membrane [29], after they have completed meiosis and formed the female pronucleus (data not shown). It is usually considered that the whole process leading to GVBD in hormonetreated oocytes can be divided into three steps. The first step is the ‘hormonedependent period’ [12, 131, which is believed to correspond to the phase of transduction of the hormonal message at the level of the plasma membrane [30, 311. During this first step, a small amount of MPF is produced under the direct influence of the hormone. In the second step the amount of MPF seems to increase, due to its autocatalytic amplification, since it sustains its biological activity following serial transfers through numerous non-hormone-treated oo-

Exo Cell Res 147 (1983)

Lithium and meiotic maturation

cytes [6]. Finally, in the third step, when the amount of MPF has reached a critical threshold value, it acts on the nuclear envelop to trigger GVBD. We do not believe that Li+ inhibits meiotic maturation simply by interfering with transduction of the hormonal message, since Li+ blocks meiotic maturation even when microinjected several minutes after the end of the hormone-dependent period. It is also unlikely that Li+ might be acting to destroy the MPF activity directly. Indeed we found that the ability of a MPF-containing crude extract to induce meiotic maturation following its microinjection into prophase-blocked oocytes was not reduced when it was first treated with 25 mM Li+. On the other hand, we eliminated the possibility that Li+ might abolish the MPF activity indirectly by first increasing the free Ca+’ concentration in cytoplasm. Our results rather suggest that Li+ interferes either with amplification or action of MPF. Indeed there was a considerable increase in the time required for GVBD to occur following transfer of MPF-containing cytoplasm into recipient oocytes, when the recipient oocytes had been previously microinjected with Lif. In some cases GVBD did not occur at all. In addition when oocytes were first treated with I-MeAde, then arrested before GVBD by subsequent injection of Li+, the cytoplasm taken from these Li+-injected oocytes did not trigger GVBD after its transfer to control untreated oocytes. Although Li+ has been shown to inhibit protein synthesis in sea urchin, it is doubtful whether the inhibition of meiosis reinitiation by Lif might result from inhibition of protein synthesis in starfish oocytes, since I-MeAde induces GVBD even in the absence of protein synthesis [12]. Moreover, amplification of MPF does not require protein synthesis in starfish oocytes [34]. Although many compounds, including alkylating agents [35], methylxanthines [13], antiproteases [36], local anesthetics [37], phenothiazines [38,39], vinblastine [38, 391, NH4Cl [14] have been shown to inhibit 1-MeAde-induced meiosis reinitiation, only nicotinamide (and its metabolite NAD) and Li+ have been shown to act by interfering either with MPF amplification or action (all the other drugs inhibit GVBD only when applied before the end of the hormone-dependent period). In both cases it was found that oocytes could be released from inhibition by raising the hormone concentration. This suggests that increasing the efficiency of MPF initiation might partially counteract inhibition of MPF amplification or action, which are believed to proceed independently of hormone action. Alternatively the possibility must be considered that the classical three-steps linear model of hormone-induced meiosis reinitiation is an over-simplification of the actual mechanisms involved in meiotic maturation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

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