Is calcium the second messenger of 1-methyladenine in meiosis reinitiation of starfish oocytes?

Experimental

Cell Research 145 (1983) 325-337

Is Calcium Reinitiation

the Second of Starfish

A. PICARD

and M. DORRE

INSERM V-249 and CNRS-CRBM, 29211 Roscoff, France

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/83/060325-13$02.00/O

Messenger Oocytes?

of I-Methyladenine

34033 Montpellier,

and Station Biologique,

in Meiosis

SUMMARY Microinjection of EGTA into prophase-blocked oocytes does not inhibit hormone-induced meiosis reinitiation, although it prevents oocyte activation by fertilization, by ionophore A23187, or by subsequent microinjection of otherwise efficient Ca*+ buffers. In contrast microinjection of Ca*+ buffers inhibits I-methyladenine-induced meiosis reinitiation. Oocytes can be released from Ca*+ inhibition by raising hormone concentration or by the subsequent transfer of cytoplasm taken from maturing oocytes. Ca’+-microinjected oocytes remain inhibited up to 1 h after microinjection, although free Ca*+ concentration comes back to its resting value less than 30 set after microinjection. Cyanide, which decreases ATP content and depresses Ca’+-pumping activity, reversibly inhibits I-methyladenine-induced meiosis reinitiation. These results do not support the hypothesis that Ca*+ is the second messenger of the hormone in meiosis reinitiation of starfish oocytes, although they support the view that elimination of Ca*+ from some component of the oocyte cortex (perhaps the plasma membrane) might be a compulsory event for transduction of the hormonal message.

It has been shown that calcium is involved in regulating a variety of enzyme systems, in most types of cell motility, and in many processes of cell activation (for review, see [l-3]). In starfish, oocytes are released from prophase block by a hormone originating from follicle cells [4], which has been identified to 1-methyladenine (1-MeAde) [51. The hormone reinitiates meiosis by interacting with stereospecific receptors [6] localized exclusively at the level of the plasma membrane and accessible to the hormone only from outside [7-91. Interaction of 1-MeAde with its receptors triggers the release of Ca*+ and a transient increase of its concentration inside starfish oocytes [ 101. Plasma membranes isolated from prophase-blocked but not from maturing oocytes exhibit a dose-dependent Ca*’ release when treated with 1-MeAde or with its biologically active structural analogs or mimetics [l 11. Increase in free Ca*+ concentration has been shown to play a key role in fertilization and parthenogenetic activation in eggs of various animals, including starfish [12-191. Evidence that Ca*+ is the intracellular effector of the cortical reaction includes suppression of the biological response in eggs injected with the Ca*+ chelator EGTA [12, 191 and its induction in eggs treated with the Ca*‘ionophore A23187 [l&18] or microinjected with Ca*+buffers of high free Ca*’ content [19]. I-MeAde has been shown to induce parthenogenetic activation when applied to fully matured oocytes [20], but not to prophase-blocked oocytes. Specificity studies suggest that this biological response results from 1-MeAde interaction with the category of receptors involved in meiosis reinitiation. Cortical reaction does not occur when the hormone is added to oocytes which have been previously microinjected with EGTA. These results support the view that 22-838331

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Ca2+ions, which are released in the cortical region of the oocytes due to hormone interaction with its membrane receptors, can trigger (or not) cortical reaction, depending on the physiological maturity of the oocytes. In many animals, activation has been shown to bring about meiosis reinitiation in prophase-blocked oocytes [21-241. In such cases activation and meiosis reinitiation appear to be closely related phenomena. In starfish, however, fully-grown prophase-blocked oocytes never reinitiate meiosis upon fertilization or ionophore A23187 addition, although they readily undergo cortical reaction under such conditions, as demonstrated by elevation of the fertilization membrane [18] and acid release [25]. Nevertheless, it has been proposed that Ca*’ might act in starfish oocytes as a second messenger of the hormone not only in parthenogenetic activation, but also in meiosis reinitiation [26, 27). The aim of the present experimental study was to examine the validity of this hypothesis.

MATERIAL

AND

METHODS

Chemicals 1 Me-Ade, Firely lantern extracts and EGTA (ethylene glycol bis(/I-amino-ethylether)-N,N’-tetraacetic acid), in its free acid form, were purchased from Sigma, USA. A 0.22 M EGTA solution was prepared by adding dropwise, under constant stirring, a 5 M KOH solution to a suspension of EGTA in distilled water. After complete solubilization, pH was 7.4. An equivalent amount of CO&a was added to part of this EGTA solution. After apparently complete solubilization, both the Ca-EGTA and the remaining EGTA solution were adjusted to 0.2 M with PIPES-KOH, pH 7.0 (PIPES, piperazineN-N’-bis(2-ethanesulfonic acid)). Final concentration of PIPES in both solutions was 10 mM and pH 7.0. The homogeneous solutions were kept at 4°C throughout the experiments, after filtration through 0.22 urn Millipore filters. Ionophore A23187 was obtained from Eli Lilly, USA, and conserved at 4°C as a 200 uM stock solution in methanol.

Handling of gametes and microinjections Fully-grown prophase-blocked oocytes of Murthasrerias glacialis were prepared free of follicle cells by washing them in Ca ‘+-free sea water (CaFSW) as described previously [8], then transferred in Millipore-filtered natural sea water. They were used for experimental l-6 h after standing, without stirring, at room temperature (about 2O’C). Dry sperm, obtained by tearing a testis in a small vessel, was kept at 4°C and used within 6 h after preparation. Fertilization was performed by adding 5 ul of a suspension of dry sperm diluted 20-fold with natural sea water to 500 pl of an oocyte suspension. Activation by ionophore A23187 was performed in the same way by adding 5 ul of a 200 uM methanolic stock solution to 500 pl of oocyte suspension. Microinjections were performed according to Hiramoto [28].

Free Ca2+ calculations In most experiments 35 pl of the different Ca 2+ buffers were microinjected into oocytes, the volume of which was about 2 nl. We indicated in table 1 the free Ca*’ concentrations which would result from a 57-fold dilution of the various 0.2 M Ca-EGTA/EGTA microinjected buffers with a 10 mM M8+ solution at the final pH 6.8. Both values were reported for Mg+ concentration and intracellular pH of unfertilized sea urchin eggs [29, 301. The buffering effect due to 0.17 mM PIPES pH 7.0, the vehicle of the Ca’+ buffers following its 57-fold dilution, was small as compared with pH buffering potential of cytoplasm, and was therefore neglected. Calculations were performed according to Portzehl et al. [31]. Association constant of EGTA for Ca*+ and Mg2+ were taken respectively as 10” and ld.2 [32]. Mg2+ was considered to reduce the apparent association constant of EGTA for Ca2 by l/l+(Mg’+)K,, where K2 is the apparent

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of starfish oocytes

constant of EGTA for Mg *+ [12]. At pH 6.8 apparent association constant of EGTA for Ca’+ was therefore calculated to be 1.23~ 106. For (Ca) totalI(EGTA) total = 1, it was calculated that free Ca*’ concentration decreased by only 0.2% when dilution was 19-fold instead of 57-fold.

Intracellular

free Ca2+ activities

They were monitored with neutral carrier-based calcium-sensitive microelectrodes, as described previously [20]. Detection limit of the microelectrodes used in this study was about 3x lo-’ M. Above 10m6 M they gave a linear response when electromotive force (emf) was plotted against pCa, with a slope of about 28.5 mV for A pCa = 1 (20°C). Time required for full response when pCa was changed from 6 to 5 was about 30 sec.

ATP measurements They were performed according to the luciferin-luciferase method [33]. Fixation of oocytes for ATP measurements was performed by injecting 1 ml of oocyte suspension (about 25 000 oocytes) in 4 ml of boiling distilled water. After 3 min boiling, the suspension was transferred in ice and then kept frozen at -20°C until ATP was measured in the supcmatant obtained from the subsequent centrifugation of the thawed suspension. It was checked by ion exchange chromatography that a trace of radiolabelled ATP added simultaneously with the oocytes in boiling distilled water did not undergo degradation during the extraction procedure.

RESULTS Attempts to inhibit I-MeAde-induced meiosis reinitiation by clamping Ca2+ concentration at a low level In a preliminary paper one of us reported that prophase-blocked starfish oocytes microinjected with EGTA failed to reinitiate meiosis when treated with 1-MeAde in artificial calcium-free sea water [ 101. Recently reinvestigation of the effects of EGTA microinjection showed, however, that prophase-blocked starfish oocytes kept in calcium-free sea water undergo irreversible cytological abnormalities, including disappearance of the nucleolus and increased thickness of the nuclear membrane, when microinjected with the Ca2+ chelator [341. Therefore the previous result must be questioned. In the present study, experiments were exclusively performed in natural sea water. The microinjected oocytes were carefully examined under light microscope and only undamaged oocytes were scored.

Table 1. Concentrations of free Ca2+ in the mixtures of 0.2 M Ca-EG1;4 and 0.2 M EGTA used for microinjection after 57-fold dilution with a 10 mM M$+ solution at the final pH 6.8 (Ca total) Mixing ratio

(EGTA total)

Free Ca*’ (PM)

l/O 10/l 3/l l/l l/3 1110

1 0.91 0.75 0.5 0.25 0.09

56 8.2 2.4 0.81 0.28 0.081

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In a typical experiment, 35 pl of 200 mM EGTA were injected in 14 prophaseblocked oocytes and 1-MeAde was added externally 1 min later, at the threshold concentration (low7 M) required for inducing meiosis reinitiation in 90% of the uninjected oocytes of this batch. This concentration did not differ whether oocytes were injected or not with 35 pl of 10 mM PIPES-KOH, pH 7.0, which was used as the vehicle of EGTA. Twelve oocytes readily underwent GVBD and resumed complete meiosis (both polar bodies were emitted). Two EGTA-injected oocytes did not undergo GVBD. However, they readily reinitiated meiosis when the hormone concentration was increased slightly: the proportion of oocytes that did not respond to 10m7 M 1-MeAde did not, therefore, differ significantly whether oocytes were injected or not with EGTA. Similar results were obtained when the time elapsed from EGTA injection to hormone addition was increased from 1 to 30 min (table 2). It has been shown that the presence of I-MeAde in the medium is required only during a well defined period of time (the hormone-dependent period) to induce meiosis reinitiation in starfish oocytes. This period cannot be reduced even by a 1 000-fold increase of the hormone concentration [6, 351. 1-MeAde was added to prophase-blocked oocytes at a threshold concentration (lo-’ M), then aliquots of the suspension were transferred as a function of time in sea water, which decreased free hormone concentration below low9 M. Less than 10% of the oocytes reinitiated meiosis when 1-MeAde was eliminated from the medium 3 min or less after its addition, whilst more than 95% of them were released from prophase block when elimination of the hormone occurred at least 4 min after its addition. Nine prophase-blocked oocytes from the same batch were first injected with 35 pl of a 200 mM EGTA solution, then transferred in sea

Table 2. Effect of EGTA microinjection Injected solution (35 PU PIPES PIPES EGTA EGTA EGTA EGTA EGTA EGTA EGTA EGTA EGTA EGTA

10 mM pH 7.0 10 mM pH 7.0 2 mM in PIPES 10 mM, pH 7.0 20 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 r&l, pH 7.0 200 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 mM, pH 7.0 200 mM in PIPES 10 mM, pH 7.0

on I-MeAde-induced

meiosis reinitiation

Time elapsed from injection to hormone application (fin)

GVBDa

lb l-26’ l-15 l-15 1 l-24 2-26 l-7 l-16 l-20 l-11 1-12

919 14114 717 13/13 616 13113 lO/lO 616 lO/lO 616 414 515

* Number of oocytes reinitiating meiosis/number of microinjected oocytes. b Oocytes were transferred in sea water containing lo-’ M I-MeAde 1 min after microinjection. ’ Oocytes were microinjected one after the other and lo-’ M 1MeAde was added at once to the group of microinjected oocytes. Therefore the delay between microinjection and hormone addition varied from 1 to several minutes.

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water containing 10 -’ M 1-MeAde; 4 min later the microinjected oocytes were transferred in hormone-free sea water. All underwent GBVD. We conclude that EGTA microinjection does not increase the duration of the hormone-dependent period. Clamping intracellular Ca2+ concentration at a low level suppresses cortical reaction inhibiting I-MeAde-induced meiosis reinitiation Although the results reported in the preceding section suggested that intracellular Ca*+ was not the effector of the hormone in inducing meiosis reinitiation, it was not demonstrated that the amount of microinjected EGTA was actually suffkient to suppress the putative intracellular Ca*+ signal resulting from hormone interaction with its membrane receptors. Prophase-blocked oocytes were injected with Ca*+ buffers of various CaEGTNEGTA ratio (table 1). When this ratio was high enough, cortical reaction occurred, although the oocytes failed to reinitiate meiosis (table 3). When microinjection was performed into the oocyte cortex, the threshold concentration of free Ca*+ required for activation was calculated to be about 7.5 PM (the threshold was increased when microinjection was deeper in cytoplasm). On the other hand, free Ca*+ concentration has been shown previously to increase by only about 0.5-l 5 uM the first 30 set which follow addition of 2~ lo-’ M lMeAde to Marthasterias glacialis immature oocytes, then to return to its basal level [lo]. Whilst oocytes injected with 35 pl of 200 mM Ca-EGTA alone (free Ca*+ concentration, 56 PM) were readily activated, cortical reaction did not occur when oocytes were first injected with the same volume of a 200 mM EGTA solution, even when EGTA was injected as much as 30 min before Ca-EGTA. Since the threshold concentration of free Ca *+ for cortical reaction to occur was Table 3. Activation rate (the number of activatedlthe total number of microinjetted) of prophase-blocked starfish oocytes microinjected with Ca-EGTAIEGTA bufers Site of microinjection

Ca2+ cont. ww

Cortex Cortex Cortex Cortex Cortex Cortex Endoplasm Endoplasm Endoplasm Endoplasm

56 8.2 7.5 6.0 4.3 2.4 56 56 56 8.1

Activation rate 7/7 (100%) 7/7 (100%) 4/4 (100%) o/3 (0%) o/5 (0%) l/6 (17%) 14/15 (93 %) 20/20 (100%) 9/18 (50%) l/8 (13 %)

a The concentration of free Ca*+ when 35 pl of Ca-EGTAIEGTA buffers containing various amounts of calcium (total EGTA, 0.2 M) is mixed with 2 nl of a 10 mM M8+ solution at the final pH 6.8 (see Materials and Methods).

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about 7.5 PM, it appears that even 30 min after its microinjection, the amount of the Ca*+-chelator in its free form was still sufficient to neutralize at least a 48.5 yM variation (56-7.5) of free Ca*+ concentration, which is by far in excess to the putative 0.5-1.5 uM Ca*’ signal resulting from 1-Me-Ade interaction with its receptors. Although oocytes injected with EGTA were prevented from activation by a subsequent Ca*’ injection (table 4), they readily underwent GVBD 13-15 min after hormone addition when 10-’ M I-MeAde was added 1 min after injection of EGTA alone. Moreover, sensitivity to 1-MeAde was not decreased, and duration of the hormone-dependent period was not increased, as already emphasized, in EGTA-injected oocytes. In another set of experiments, oocytes were fertilized or treated with 2 uM ionophore A23187, at various times after the microinjection of 35 pl of 0.2 M EGTA. Thirty seconds after EGTA microinjection, 10m7 M 1-MeAde was added in the medium. All oocytes underwent GVBD 13-15 min after hormone addition and complete meiosis later. In contrast, cortical reaction never occurred, even when fertilization or ionophore application was performed after GVBD. The same result was obtained when fertilization and ionophore application were performed before hormone addition, but after EGTA injection (table 5, fig. 1). We conclude that failure of EGTA to inhibit meiosis reinitiation is not due to insufficient amount of the microinjected chelator. Injecting Ca*+ buffers with high free Ca*+ content inhibits I-MeAde-induced meiosis reinitiation Oocytes were injected with 35 pl of Ca *+ buffers of various Ca-EGTNEGTA ratio (total EGTA concentration, 200 mM) then 1-MeAde was added at its lowest concentration for inducing meiosis in more than 95 % of the uninjected oocytes. The percent of GVBD was estimated 1 h later. Injection of Ca*’ buffers was found to inhibit meiosis reinitiation, and the extent of inhibition increased with free Ca*+concentration. Oocytes could be released from inhibition either by raising hormone concentration (table 6) or by transferring 150 pl of cytoplasm

Table 4. Suppression of the cortical reaction due to Ca-EGTA microinjection a previous microinjection of EGlX EGTA-injected

oocytes

by

Control oocytes

At” (min)

Final Ca2+ cont. (PM) b

Activation rate

Ca” cone (Of) ’

Activation rate

10 34

0.82 0.82

O/6 o/10

56 56

717 8/8

a Time elapsed from the first to the second injection. In EGTA-injected oocytes 35 pl of 200 mM EGTA were injected frst, and 35 pl of 200 mM Ca-EGTA later. In control oocytes, 35 pl of 10 mM PIPES pH 7.0 (the vehicle of EGTA) was injected first and 35 pl of 200 mM Ca-EGTA subsequently. b Calculated as for tables 1 and 3.

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taken after GVBD from maturing oocytes and containing MPF, the maturationpromoting factor [36-381. Since most oocytes did not escape spontaneously the Cazf-induced inhibition, even several hours after microinjection, the results suggest that the Ca2’ buffers perhaps acted by clamping intracellular free Ca*+ at a high level. Nevertheless, we observed with Ca2+ -sensitive microelectrodes that intracellular Ca2+ activity did not increase, except very slightly during the course of microinjection, when as much as 35 pl of 0.2 M Ca-EGTA (free Ca2+ content, 56 yM) were injected into prophase-blocked oocytes of Marthasterias glacialis (fig. 2A). Similar results were obtained in Asterias rubens (data not shown). This result cannot be accounted for by insufficient sensitivity of the Ca*+-sensitive microelectrodes in the range of intracellular Ca*+ activity, since emf still increased by a few mV when intracellular Ca*+ was clamped at a still lower level by EGTA microinjection (fig. 2A). The above result suggested that a potent Ca2+-ATPase was at work to pump Ca*+ out of cytosol. Therefore we tried to depress the Ca*+-sequestering machinery by poisoning the oocytes with cyanide. After 45 min treatment with 100 uM KCN, the intracellular ATP content decreased from 2.94 to 1.56 mM. Under such conditions, a significant increase of the intracellular CaZf activity was observed when 30 pl of 0.2 M Ca-EGTA were injected. After a 2 h treatment with 100 uM KCN, the background Ca2’ activity increased significantly in the oocytes, and it further increased considerably following Ca-EGTA microinjection (fig. 2B). Finally, a 45 min pretreatment with 100 PM KCN was found to increase the dose of hormone required for meiosis reinitiation in prophase-blocked oocytes. The hormone concentration required for 50% of the oocytes to undergo Table 5. Clamping intracellular Ca*+ concentration cortical reaction induced by fertilization or ionophore I-MeAde-induced meiosis reinitiation Activation stimulus

A tie (min)

A t2’ (min)

Activation rate

Fertilization Fertilization Fertilization Fertilization Fertilization Fertilization Fertilization Fertilization Fertilization A23187 A23187

2 1 1.5 3 4 5 7 9 11 2-10 4-12

3 0.5 1 0.8 0.8 0.8 0.8 0.8 0.8 l-9 3-l 1

o/3 o/2 o/2 o/3 O/5 o/2 o/2 o/3 O/5 O/5 o/5

at a low level suppresses A23187 without inhibiting

GVBD 313 212 212 313

515 212 212 313 515 515 515

a Time elapsed from EGTA microinjection (35 pl, 200 mM) to either fertilization or addition of 2 uM ionophore k23 187. b Time elapsed from EGTA microinjection to the addition of 8~ 10-s M I-MeAde (this was the lowest hormone concentration required for inducing meiosis reinitiation in more than 95% of the uninjected oocytes).

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Fig. 1. Suppression of the cortical reaction by a previous microinjection of EGTA does not inhibit hormone-induced meiosis reinitiation. The activation stimulus used to trigger the cortical reaction was either ionophore A23187 (A,, AZ, B,, IQ, fertilization (C,, CZ, Dt, DZ), or microinjection of CaEGTA (Et, Ez, F,, F2). 35 pl of a 200 mM EGTA solution was microinjected (Al, B,, C,, D,, E,, F1) or not (AZ, &, Ca, D2, E2, Fz) 3 min before application of the activation stimulus. The hormone was added (final cont. 2x lo-rM) 5 min after application of the activation stimulus. Al, A*, Micrographs taken 5 after application of ionophore A23187 (thus simultaneously with hormone addition). The two silicon oil droplets which travel with the microinjected sample of EGTA are visible in oocyte 2. B,, &, the same oocytes 30 min later. GVBD occurred in both oocytes, although EGTA microinjection suppressed cortical reaction in oocyte 1. C,, Cz respectively as A,, AZ and B,, LIZ excepted that fertilization was used as the activation stimulus instead of ionophore. El, E2, respectively as A,, AZ and Br, B2 excepted that microinjection of 35 pl of a 200 mM Ca-EGTA solution was used as the activation stimulus (thus four silicon oil droplets are visible in oocyte 1). A,, A*, X158.

GVBD were respectively 0.02 and 1 uM in control and in KCN-treated oocytes. Inhibition was more pronounced when the duration of the cyanide pretreatment was increased. DISCUSSION In terms of the classical second messenger model, recognition of a specific extracellular first messenger involves its interaction with cell surface receptors;

Res 145 (1983)

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Ca2+ and meiosis reinitiation

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the resulting transduction of this messageleads to the generation or to the release of the second messenger that is transmitted by diffusion within the cytosol, where it is recognized by regulatory subunits of intracellular receptors which in turn trigger cell sequestration of the second messenger [39]. By analogy to CAMP, it has been assumed that Ca2+ acts as an intracellular second messenger in many Ca2+-mediated processes.. Calmodulin has been proposed as the universal intracellular calcium receptor involved in the regulation of such Ca’+-mediated processes [ 1, 2, 401. In starfish it has been suggested that Ca2+ might be the second messenger of lMeAde in hormone-induced meiosis reinitiation, This hypothesis is mainly substantiated by the demonstration that l-MeAde receptors are localized on the plasma membrane and by the finding that prophase-blocked oocytes injected with the Ca2+-sensitive photoprotein aequorin emit light less than 2 s following external application of the hormone. Change in intracellular free Ca2+ is, however, restricted to the first 30 s which follow 1-MeAde addition; then the Ca2+ concentration returns to the level of unstimulated oocytes [IO]. In contrast, presence of the hormone is required during several minutes for cell machinery to be irreversibly released from prophase block [35]. Ionophore A23187 and fertilization, which increase intracellular Ca2+ much more than the hormone, do not reinitiate meiosis in starfish oocytes [lo, 18, 251. The same negative result was obtained, even in Na’+-free sea water,

Table 6. Inhibition

of I-MeAde-induced meiosis reinitiation by the microinjection of 35 pl of Ca-EGWEGTA buffers (total EGTA, 200 mM) containing high levels of free calcium Ca2+ buffer-injected

Expt no. 1 2

I-MeAde concentration CM) 4X 10-8 5x10-7 4x 10-8 8x lo-’ 1x10-7 5x10-7

I&

0 tOW (EGTA total)

oocytes GVBD’

Control oocytes b GVBD

l/O l/O

o/9 (0 %) 8l9 (88 %)

9/10 (90%) 9/10 (90%)

l/O l/O l/O l/O

o/10 (0 %) l/8 (13 %) 2/14 (14%) 1304 (93 %)

5125 (20%) 23/25 (92 %) 24/25 (%%) 25/25 (100%)

lo/12 (83 %) 8x10-* l/O l/10 (10%) 7/10 (70 %) 11111 (100%) 1x10-7 l/O 10110 (100%) 1:6x10-’ l/O lO/lO (100%) 8x10-* 8/8 (100%) 9/10 (90 %) 4 O/l 8x10-* 7/9 (78 %) 9110 (90%) l/3 4/6 (67 %) lO/lO (100%) 8x10-’ l/l 2/6 (33 %) lO/lO (100%) 8x lo-’ 10/l 9/10 (90%) 1115 (7 %) 8x lo-’ l/O ’ Number of oocytes reinitiating meiosis/total number of microinjected oocytes. b Control oocytes were injected with 35 pl of 10 mM PIPES pH 7.0, the vehicle of the Ca2+ buffers. No difference in sensitivity to 1-MeAde was observed, whether oocytes were injected or not with PIPES.

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lmin -

Fig. 2. Original records of intracellular Ca*+ activity (upper trace, ECa) and membrane potential (lower trace, Vm) in oocytes poisoned (B) or not (A) with 100 PM KCN. Oocytes were impaled successively with two microelectrodes (a conventional KCl-filled microelectrode and a Ca*+sensitive microelectrode) and one micropipet filled with silicon oil which contained at the tip the indicated volumes of either a 200 mM EGTA solution or a 200 mM Ca-EGTA solution. Both microelectrodes were first introduced in sea water where the oocytes were bathing (reference potential). Arrows I, The conventional microelectrode was first inserted, therefore the Eta trace was deflected upward out the scale, because the Eta record is the difference signal between the Ca*‘sensitive and the conventional microelectrodes. The Ca*+ -sensitive electrode was inserted next (arrows 2), then the micropipet (arrow P). Microinjections were performed about 1 min later. (A, left) Effect of the injection of either 30 pl of EGTA or 30 pl of Ca-EGTA on Eta in control prophaseblocked oocytes. (B, right) Phophase-blocked oocytes were first poisoned with 100 PM KCN during 45 min (first record) or 2 h (second record), before injecting the indicated amount of the Ca*+ buffer.

when continuous depolarizing current pulses, which causes Ca’+-Na+ action potentials and thus an influx of Ca2+ ions, were applied to prophase-blocked oocytes [41]. In addition we show in the present work that microinjection of Ca” buffers containing high amounts of free Ca2+ triggers the cortical rection, but not meiosis reinitiation. Taken together, these results show that other factors unrelated to Ca” release must also play an essential role in meiosis reinitiation of starfish oocytes , even if Ca*’ acts as a compulsory second messenger of the hormone.

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Ca2+ and meiosis reinitiation

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Even with this restriction, the second messenger hypothesis predicts that suppressing the transient increase of Ca2+ which follows hormone addition must also suppress meiosis reinitiation. In the present study we show, however, that microinjection of EGTA, which prevents prophase-blocked oocytes to be activated by fertilization, by ionophore A23187 or by the microinjection of Ca2+ buffers, does not inhibit I-MeAde-induced meiosis reinitiation. In fact, the sensitivity to the hormone is not decreased and the duration of the hormone-dependent period is not increased in EGTA-microinjected oocytes, with respect to control uninjected oocytes. Both meiosis reinitiation and activation involve the transient increase of free Ca2+ mainly, if not exclusively, in the cortical region of the oocyte [3, 11, 12, 19, 451. Free Ca2+ concentration has been shown to increase by about 0.5-1.5 uM following 2~ lo-’ M I-MeAde addition [IO]. Although the extent of this transient free Ca2+ peak increases when hormone concentration is raised from 1O-9 to lo-’ M [ll], it always remains much smaller than the transient free Ca2+ peak which occurs when oocytes are activated by fertilization or ionophore application. EGTA, which was shown in present work to neutralize a 48 pM variation of the free Ca2+ concentration even as late as 30 min after its microinjection, would therefore be expected to prevent free Ca2+ concentration to increase significantly in oocytes treated with a threshold concentration of 1-MeAde. We believe that these results rule out the hypothesis that Ca2+ might act as a second messenger of the hormone in meiosis reinitiation. This opinion is also supported by recent reports that requirement for I-MeAde and sensitivity to the hormone were modified neither by the microinjection of Ca2+-calmodulin [42] nor by the microinjection of anticalmodulin drugs and antibodies [43]. It has been shown, however, that drugs which suppress the release of Ca2+ or decrease it under a threshold value either in living oocytes or from plasma membrane-rich fractions abolish meiosis reinitiation [ 11, 441. Therefore the presently available experimental results strongly support the view that elimination of Ca2+ from some component of the oocyte cortex (perhaps the plasma membrane) is an essential event for meiosis reinitiation [42, 451, although the released Ca2+ does not act as a second messenger of the hormone in this biological response. In contrast Ca2+ is certainly the intracellular effector of 1-MeAde in parthenogenetic activation of fully-matured starfish oocytes 1201. According to this interpretation, treatments which increase the Ca*+ content in oocyte cortex would be expected to increase also the dose of hormone required to induce meiosis reinitiation, even if free Ca2+ comes back rapidly to its basal -sequestering machinery which operates in starfish level due to the potent Ca2+ oocytes as in most cells [46-48]. We found indeed in the present study that oocyte poisoning with cyanide, which decreases ATP content and depresses Ca2+pumping activity, reversibly inhibits I-MeAde-induced meiosis reinitiation. Dinitrophenol has also been shown to have the same effect [49]. Moreover, oocytes microinjected with Ca2+ buffers containing high levels of free Ca2+ require a much higher dose of hormone than control uninjected oocytes (or than oocytes Ca2’injected with the vehicle of the Ca2+ buffers) for meiosis reinitiation. microinjected oocytes remain inhibited even 1 h after microinjection, although

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free Ca2+ concentration comes back to its resting value less than 30 s after microinjection. We reported elsewhere that short preincubation of starfish homogenates, but not 105000 g supernatants, in the presence of Ca2+, with or without inhibitors of Ca*+-dependent proteases, strongly inhibits protein kinase activity, even if the protein kinase assay itself is performed in the presence of excess EGTA [45, 501. Since the rate of [32P]phosphate incorporation into proteins increases at least IO-20-fold following I-MeAde action [51, 421, the possibility must be considered that increased Ca2+ might also be detrimental to protein phosphorylation in the intact starfish oocyte.

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Res 145 (1983)

Exp Cell Res 145 (1983)

Cu2+ and meiosis reinitiation

of starfish oocytes

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