- Email: [email protected]
Hormonal control of meiosis
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 152.0251$02.00/0
Experimental Cell Research 115 (1978) 251-260
HORMONAL
CONTROL
OF MEIOSIS
In vitro Induced Release of Calcium Ions from the Plasma Membrane in Star-sh Oocytes M. DORRE, M. MOREAU and P. GUERRIER Station Biologique,
F-29211 Roscoff,
France
SUMMARY Using the photoprotein aequorin as a marker, we found that the Ca*+ release, which we demonstrated to occur from the inner part of the plasma membrane after hormonal stimulation of intact starfish oocytes can also be recorded in vitro. Our acellular system was prepared from cortices of Murthasterias glacialis oocytes isolated in Ca*+-free medium and extracted with Triton X-100. At O-PC, the 100000 g pellet obtained from this plasma membrane-rich extract responds in less than 0.1 set to the hormone I-methyladenine, its biological active analogues and mimetics, whereas supematant exhibits a quite lower activity which is no longer present in the head fraction recovered after dialysis through Diatlo UM 2 filters. The in vitro calcium response is competitively inhibited by various agents which, in the same concentration range, act on meiosis reinitiation and free calcium release by the living oocyte. These and previous data are further discussed taking into account the overall chain of events which leads to the release of meiosis inhibition.
In starfish, full grown oocytes are arrested at the prophase stage of meiosis. Meiosis is reinitiated by a hormone produced by the follicle cells [l-5]. The hormone has been identified as I-methyladenine ( I-MeAde) [6-71. It has been demonstrated that lmethyladenine controls meiosis by way of stereospecific receptors localized on the oocyte plasma membrane [8-l 11. Recently, we found that 1-MeAde induces the release of intracellular calcium in Murthasterius glacialis intact oocytes, which is essential for meiosis reinitiation inasmuch as any treatment which suppresses it or decreases it under a threshold value abolishes meiosis reinitiation [12-131. We present here new data showing that this release can be recorded in vitro as an immediate effect of the concentration-dependent and specific interaction of the hormone with a plasma mem-
brane-rich fraction prepared from isolated starfish oocytes cortices.
MATERIAL
AND METHODS
Fractionation procedure glacialis oocytes were prepared free of follicle cells [9] and processed according to the modification of Sakai’s procedure described by &terrier 1141to obtain isolated cortices: oocytes were collected by gentle centrifugation, washed rapidly with NaCl 0.53 M-Tris maleate 0.05 M pH 8.2 (no EDTA was added), which was cleared of traces of calcium through a Chelex 100 column (bed height 30 cm, i.d. 4 cm), and submitted to 10 strokes of a hand homogeniser fitted teflon pestle in ice cold buffer. A pellet was obtained after 1 min centrifugation at 1000 g. The first supematant was collected (endoplasmic fraction). It contained 98% of oocytes proteins and most mitochondria. The 1000 g pellet was washed once again with the same buffer to obtain isolated cortices, which contain the microvilli-rich peripheral envelopes and no characterized mitochondria as shown by electron microscopy. Cortices were resuspended in the same buffer containing 0.2% Triton X-100 and very slowly
Marthasterias
Exp Cell Res 115 (1978)
252
Dorte, Moreau and Guerrier Follicle
cells-free
Potter (buffer
Fractionation of lo5 g supernatant obtained from the plasma membranerich fraction
oocfles
homogeneisation
A 0.53M NaCUL05M passed through a chelex
30 * centrifugatton
Tris-maleate x 100 col”mn
at 1000
pH8.2)
Ultrafiltration of the lo5 g supematant (230 pg protein/ ml) though Diaflo UM2 filters were performed at 4 “C in an Amicon device under 2.1 Atm (30 psi) N2. 500 ~1 dialysates contained 25 pg protein by ml.
g
pjjq+y-q
washed
with
buffer
A
pz+ resuspended for 30 mn at 0°C in 0.2 % Triton in buffer A 5 mn centrifugation
at 1000
g
p(pi/izpJ
Electron microscopy The pellets were fixed in 6% glutaraldehyde, 0.2 M cacodylate buffer, pH 7.4,3.5% NaCI, post-fixed with 1% osmium, 0.2 M cacodylate, 5% NaCl and embedded in Spurr medium. Ultrathin sections were prepared with an LRB ultramicrotome and stained with uranyl acetate and lead citrate.
Handling 40000 10’9
rpm SW 50.145 /\ pellet
In”
10” g supernatant
Fig. 1. Procedure for obtaining the plasma membranerich fraction.
shaken at 0 “C for 30 min before being centrifuged again at 1000 g for 5 min. Aggregated ghosts were recovered in the pellet. The clear supematant, which contained about 40% of the total proteins of the isolated cortices, was collected (“Plasma membrane-rich fraction”). A lo5 g pellet and a 10s g supematant were prepared from this fraction after 45 min centrifugation at 40000 rpm with an SW50.1 rotor in a Beckman L5-75 ultracentrifuge. The protein content was 30-40 fold higher in the supematant than in the pellet. Fig. 1 depicts the flow diagram for preparaing the different subcellular fractions.
of aequorin
Aequorin, grade I, purchased from Sigma, was puritied by chromatography through small G25 Sephadex columns equilibrated with 1 pm M EDTA., 10 mM sodium acetate, pH 6.2. 50 /~1aequorin solutions were put in small plastic preparation dishes mounted directly on the photomultiplier enclosed in a light-tight cage. Subcellular fractions (500 J) and chemicals (50 ~1) were injected into the aequorin solution by remote control through a plastic syringe. Flash emissions were recorded as already described [12]. From 0.05 to 0.4 nA, increases of anode current were found to be proportional to the quantities of Ca*+ introduced or released in the preparation dish. Although we always worked in this range, we found it better to express our results in arbitrary units since calibrations performed as indicated on fig. 3A showed that the light response (anode current deviation) recorded after adding a known amount of Ca*+ to the vessel may vary from one batch of aequorin to the other.
RESULTS Enzymes assays The reaction mixture for 5’-nucleotidase assay was made from 50 ~1 &Y-AMP low3 M (0.1 &i), 100 ~1 Tris lo-’ M, pH 8.5, MgCI, lo-* M and 50 ~1 sub-cellular fraction. 50 ~1 aliquots were collected as a function of time, boiled for 3 min after injection into 150~1 distilled water, and passed through a small Pasteur pipette filled with 1 ml DEAE Sephadex. The pipette was rinsed with 2 ml of distilled water and the eluate containing adenosine (and eventually adenine formed by nucleoside hydrolase interfering activity) as the reaction product, was counted for radioactivity. The reaction mixture for alkaline nitrophenylphosphatase was made from 200 ~1 paranitrophenyl-phosphate lo-* M, 200 ~1 Tris 0.2 M MgCI,, 5 mM, pH 9, and 200 ~1 subcellular fraction. Release of nitrophenol was measured at 420 nm. Subcellular fractions for determination of enzymic activities contained 0.5-2 mg proteins/ml. Exp Cell Res 115 (1978)
Fig. 2A shows a phase contrast micrography of cortices isolated from Murthasterius glacialis oocytes. Such isolated cortices include vitelline membrane, plasma membrane and the 2-5 pm superficial layer of the oocyte . This cortical ghost first fraction which was found to possess adenylate cyclase and phosphodiesterase activity (cryptic and not cryptic), readily responded to hormonal stimulation by releasing free calcium in the medium. Controls were run using aequorin + ghosts or aequorin + I-MeAde prepared
In vitro release of Ca2+
Fig. 2. (A) Isolated cortical ghosts obtained by homogenization in the extraction medium. The bar represents 20 PM. (B) Electron micrograph of the 1oJ g pellet fraction obtained from the low speed supematant after extracting the cortical ghosts 30 min at 0 “C with 0.2% Triton X-100. The bar represents 0.5 pm.
in the extraction medium which always gave negative results. Mild treatment with diluted detergents releases a fraction containing a homogeneous population of membrane vesicles ranging from 0.1 to 0.4 pm in diameter which is re-
253
covered in the 105g pellet (fig. 2B). Some sections demonstrated, however, the presence of scarce contaminating electron dense and fibrous material which is likely to derive from cortical granules which may have been destroyed during preparation in our EDTA- and EGTA-free medium [IS]. Significant S-nucleotidase and alkaline nitrophenylphosphatase activities were found to be associated with the membrane vesicles (table 1). Since the enzymes have been demonstrated to be specific plasma membrane markers [ 16-211 the fraction containing the membrane vesicles was referred as the “plasma membrane-rich fraction” in this text, although most of its proteins remains in the lo5 g supernatant after centrifugation. Fig. 3B shows an original record of the response displayed on the oscilloscope when 2x lo-’ M I-MeAde was added to the plasma membrane-rich fraction in a typical experiment. Less than 0.1 set after hormone addition, the anode current increased from 0 to 0.5 nA, which corresponds to an increase of free Ca2+ions in the vessel from 0 to about 2x lo-l2 ion g (fig. 3A). Ten seconds after hormone addition, free Ca2+level had already come back nearly to zero. Table 2 shows that only those analogues of 1-MeAde which can replace the hormone in triggering meiosis reinitiation of intact
Table 1. Specific activities of two plasma membrane marker enzymes in subcellular fractions from Marthasterias glacialis oocytes a Isolated cortices Plasma membrane rich fraction Enzymes
Endoplasm
Cortical pellet
Total
105g pellet
l@g supematant
5’ nucleotidase Alkaline nitrophenylphosphatase
2 0.1
35.2 0.75
2 0.1
44 0.9
1 0.02
a Expressed as pmoles substrate transformed/h/mg proteins. Exp Cell Res I I5 ( 1978)
254
Dorke, Moreau and Guerrier
Fig. .?. Light responses recorded on the oscilloscope. (A) Calibration: lo-‘* ion g Caz+ (0.05 ml of a 20 nM
Ca*+ solution) were added to 0.4 ml of the extraction medium + 0.03 ml of aequorin solution. Identical value was recorded when extraction medium was substituted by the plasma membrane-rich fraction used in B and C, which reoresented extracted cortices from about 2X lo5 oocytes; (8) response following stimulation with 2X lOA7M I-MeAde (final concentration): (C) response obtained with 10 mM DTT (final cdncentration). Vertical and horizontal bars correspond respectively to 0.12 nA anode current and to 12 sec.
oocytes are able to release Ca*+ ions from the plasma membrane-rich fraction. Moreover, fig. 3C shows that addition of dithiothreitol (DTT), which mimics I-MeAde in inducing meiosis reinitiation [22], increasing protein-SH in the oocyte cortex [23] and stimulating protein phosphorylation [24] also results in an important release of Ca2+, although the quite different time course of CaZf release suggests that DTT does not act as specifically as the hormone and releases also Caz+ from membranes sites which are not essential for meiosis reinitiation. Fig. 4 shows that 1-MeAde triggers only a very small release of Ca2+ ions from the endoplasmic fraction, in contrast with the two fractions obtained from isolated cortices. Moreover, it makes clear that Ca2+ release is higher from the plasma membrane-rich fraction than from the residual cortical pellet. The lo5 g supernatant obtained from the plasma membrane-rich fraction, which contains most proteins, only slightly releases Ca2+ions following lMeAde addition, in contrast with the lo5 g pellet, which contains the plasma memExp Cell Res I15 (1978)
brane vesicles. No activity is found in the head fraction recovered after dialysis on Diaflo UM2 filters. Fig. 5A shows the response of the same batch of plasma membrane-rich fraction to stepwise addition of I-MeAde. Ca*+ release is already noticeable with 2~ low9 M l-MeAde and stepwise increase of the 1-MeAde concentration from 2x lo+ M to 2x 10e6M enhances the amount of Caz+ ions released after each hormone addition. However, repeated hormone additions at high concentration exhaust the ability of the plasma membrane-rich fraction to release further Ca2+ions, although less than 5 % of the total membrane-bound Cal+ has been previously released (as shown after injuring the plasma membrane-rich fraction with 20 % Triton X100). We have shown that methylxanthines competitively inhibit meiosis reinitiation and stimulation of protein phosphorylation induced by both 1-MeAde and DTT [lo, 241. Fig. SA shows that theophyllin (Sigma) also competitively inhibits the hormone-induced Ca2+ release from the plasma mem-
Table 2. Ability of various structural analoguesa to replace I-MeAde in triggering meiosis reinitiation and calcium release from plasma membrane-rich fraction Compounds 1-Methyladenine I-Ethyladenine 1Benzyladenine I-Isopentenyladenine 1,N, 6Dibenzyladenine
Meiosis reinitiation
Calcium release
Active
Active
Inactive
Inactive
I
~:$:“,:~~~P~~~nthine I-Methyladenosine
1~o$r$o$p~a$e-5’1,7-Dimethyladenine
I
n All compounds were used at the concentration to-6M.
In vitro release of Cazf
A
B
255
C
Fig. 4. Abscissa: subcellular fractions tested for Ca*+ release after addition of 10-r M I-MeAde; ordinate: amount of Cal+ ions released per 138 pg of proteins (arbitrary units proportionally to anode current). Isolated cortices were fractionated into a plasma membrane-rich fraction (A) and a residual cortical pellet (B) according to the procedure described in fig. 1. Fraction A and endoplasm (C) were tested for Cal+ release before (Z’) or after subfractionation into a 105g pellet (P) and the corresponding supematant (S). lMeAde 10e6M in 0.05 ml was added to a 0.5 ml assay containing respectively 102 pg (A, S); 92 ,ug (A, P); 138 pg (A, T); 120 pg (B) and 70 pg proteins (C, 7’). Original recorded values for these concentrations appear in black, hatched area represents adjustment up to 138pg proteins.
brane-rich fraction, Essentially identical results were obtained with caffeine (Sigma) or when DTT was substituted for I-MeAde. Similar inhibitions of 1-MeAde induced Ca2+ release from plasma membrane-rich fractions were recorded with various agents which block meiosis reinitiation and free calcium release in the living oocyte [12, 241 such as 10 mM MnCl,, 0.5 mM D 600 (Knoll), 5 mM procaine hydrochloride (Merk) or N-ethyl maleimide 0.1 mM (Sigma) and Emetine 400 pg/ml (Sigma). In all cases inhibition of Ca2+ release was released by raising 1-MeAde concentration. Competitive inhibition of the calcium and biological responses occurred in the same range of concentrations, both in vivo and in vitro [25]. Plasma membrane-rich fractions were now prepared from oocytes which had been
Fig. 5. Abscissa: I-MeAde concentrations; ordinate: amounts of Ca*+ ions released per mg of proteins (in arbitrary units proportionally to anode currents). Effect of stepwise increases of I-MeAde concentration on the amount of Cazf ions released from the plasma membrane-rich fraction. (A) A plasma membrane-rich fraction prepared from prophase-blocked oocytes (before hormonal treatment) was divided into two batches, one of which received theophyllin to the final concentration 5x 10-s M, the other serving as control. I-MeAde was added stepwise to the final concentration indicated (from 2x lO-9 M to 4x lOA M for the control; from 2x 10m9M to 2x lOmEM for the theophyllin-treated batch. The height of each block refers to the amount of CaZ+ ions released in the control batch, the height of the black area to the amount of CaZ+ released in the theophyllin treated batch. (B) Plasma membrane-rich fraction prepared from oocytes which had been first treated for 8 min with 2x 10-OM I-MeAde: stepwise addition of I-MeAde to the final cont. 2X 10-r M. 5x 10-r M and low6M.
already treated for 8 min with 2X lo-” M lMeAde and then washed free of hormone. Fig. 5B shows that addition of 2~ lo-’ M 1-MeAde elicits only a very small residual release of Ca2+ ions, which cannot be increased by further hormone application.
DISCUSSION The present results show that a specific release of free Ca2+ions from plasma membrane-rich vesicles obtained from isolated cortices is triggered by 1-MeAde, its active analogues and its mimetic agent, DTT. It is very likely that the hormone cannot release Ca2+ ions from mitochondria or endoplasmic reticulum which constitute the main Exp CdRes
115 (1978)
256
0’
Dot-de, Moreau and Guerrier
’ 10-3
I 10-z
I 10-l
, 1
1
I
10
100
I
Fig. 6. Abscissa: Hormone concentration in micromoles; ordinate: receptors occupancy. Schematic representation of receptor occupancy as derived from the amount of Ca*+ released in intact oocytes treated with I-MeAde. The curve represents the quantities of light emitted by aequorin-injected oocytes following addition of I-MeAde at various concentrations [12]. Levels of receptors occupancy for maximal values of the various elementary responses are shown. a, Threshold for in vivo and in vitro calcium release; b, saturation for Na+, K+-dependant ATPase; c, saturation for stimulation of protein phosphorylation and for meiosis reinitiation.
membrane systems of the endoplasmic fraction, since the very small Ca2+ release found with this fraction could be accounted for by the contamination by some peripheric membrane fragments which would be expected. Since removal of the vitelline membrane neither reduces the calcium release induced by the hormone in intact oocytes [12] nor modifies the biological responses [26, 271, the present results strongly support the view that only the plasma membrane releases Ca2+following hormone application. This agrees very well with our previous studies which demonstrated the absence of intracellular receptors for lMeAde recognition [ 1l] and established that I-MeAde receptors are exclusively localized at the level of the plasma membranes [8, 91. In this study, we found that a plasma membrane-rich fraction extracted Exp Cell Res 115 (1978)
from about 2 x lo5 oocytes released 2 x lo-l2 Ca2+ ion g. This corresponds to a basal release of about lo-l7 ions by oocyte cortex, which would account for a 5 nM variation if adjusted to the 1760picoliters oocyte volume. When compared with the micromolar range variation found in vivo [12, 131, this points to a 1% recovery, following our procedure. Since higher values were also obtained in other experiments (up to 10F” ion g), it seems likely that Ca2+ binding sites are more readily accessible to hormone stimulation in the in vitro than in the in vivo conditions. Another possibility may be worth considering, however: that our preparation has been complemented with membranes originating from cortical granules lysed during triton extraction and which may eventually respond to hormonal stimulation. This makes little difference, however, since one can not argue that our system has become by the fact highly different from the membrane 1-MeAde interacts with normally. We found indeed that germinal vesicle-bearing oocytes, which were preactivated by ionophore A 23 187 and have released cortical granules matrix [13], readily responded to the hormone and were even more sensitive than untreated ones. In these conditions, 50% meiosis was reached, using 6x lo+? M 1-MeAde, instead of the 1.4~ 10e7 M concentration required with untreated controls from the same batch. Ca2+release from the plasma membrane, stimulation of the ouabain sensitive Na+/K+ pump [28], stimulation of protein phosphorylation [24] and meiosis reinitiation [lo] share in common the same specificity. The only active structural analogs of 1-MeAde are Nl-substituted adenines; activity is conserved or even increased when the methyl group is replaced by larger substituent groups; activity is lost when N l-substituted
In vitro release of Ca2’
257
adenines are further substituted at the N7 be considered for such a process: these may or at the N9 position, whereas it is con- be a direct displacement by the hormone of served in Nl, N6 disubstituted adenines. Ca2+ normally bound to the receptor site. This strongly supports the view that all Alternatively, the effect of the hormone these biological effects are consequences of may be considered as an allosteric linkage I-MeAde interaction with the same cate- with the hormone serving (when bound to gory of plasma membrane receptors. Ob- its specific recognition site) to reduce the tention of similar concentration dependen- affinity of the Ca2+ binding site. This latter cies for binding and biological response has mechanism, which assumes two different often been considered as evidence that the sites on the plasma membrane for both redetected binding sites were the receptors in- cognition function and effector function volved in the biological response, although (calcium release) seems to be more plausithe molecular mechanism involved in the ble since previous studies based on microtransduction was unknown. However, our injection techniques [8, 111 or competitive system clearly shows that the threshold inhibition of labelled I-MeAde uptake [9] value of hormone concentration required to have established that the hormone receptor obtain a biological effect may depend on the is accessible to 1-MeAde from the outside nature of the biological effect under inves- of the cell and not from the inside, whereas tigation: maximal stimulation of the Na+/K+ Ca2+ release occurs exclusively on the inpump is already obtained with nanomolar side of the plasma membrane [12]. The rehormones concentrations [28], maximal ceptor and effector function may reside at stimulation of protein phosphorylation and two different sites of the same molecule, or meiosis reinitiation occur in the 0.1-0.2 PM more likely, reside in separate molecules range [lo, 241 whereas maximal Ca2+ re- which interact directly or indirectly in the lease is reached only with micromolar hor- fluid mosaic membrane. We showed previmone concentrations. According to the oc- ously that Ca2+ release already occurs l-2 cupany theory [34, 351, this would imply set after application of 1-MeAde to intact that a maximal effect at the level of the oocytes [12, 131;now we found that isolated Na+/K+ pump is achieved with only a mi- plasma membranes already release Ca2+ nority of receptors occupied. The additional ions less than 0.1 set after 2~ lo-’ M l“spare” receptors [29] are not inactive re- MeAde addition at 4°C. On the other hand ceptors since the other biological responses suppression of calcium release by methylrequire greater level of receptors occupan- xanthines, MnCl,, D600 or procaine supcy. Even meiosis reinitiation, however, presses both metabolic activation [24, 281 which is the most integrated biological re- and meiosis reinitiation [lo, 12, 131. These sponse to the hormone, does not require full findings strongly support the view that Ca2+ occupancy of the receptors. Fig. 6 gives a release from the plasma membrane into the schematic picture of receptors occupancy cortical region of the oocyte is the first step for the various responses of the oocytes to in the sequence of events leading to metabolic activation and meiosis reinitiation. It hormone application. Inasmuch as both interaction of 1-MeAde is very likely that loss of Ca2+ from the with its stereospecific receptor and release plasma membrane is not by itself sufficient of Ca2+ions occur at the level of the plasma to initiate this sequence and that the free membrane, two possible mechanisms may Ca2+ concentration must actually increase Exp Cell Res I IS f 1978)
258
Dot-&e. Moreau and Guerrier
transiently (probably only in the oocyte cortex) since EGTA, which would not be expected to inhibit Ca2+ ions release, suppresses meiosis reinitiation if it is injected into intact oocytes before development of the free Ca2+peak, i.e. less than 10 set after hormone addition [ 121. Protein phosphorylation is stimulated more rapidly (less than 2 min after hormone addition) and extensively in the cortex of the oocyte than in the endoplasm, suggesting that phosphorylation of some cortical proteins might be a major event in meiosis reinitiation [24]. This agrees with our results which suggest that the target for Ca2+-induced metabolic activation must be located in the vicinity of the plasma membrane, although the exact mechanism still remains to be elucidated. The finding by Kishimoto & Kanatani that the protein-SH content increases in the cortex following hormone addition points in the same direction [22]. We showed elsewhere that the presence of I-MeAde in the range 0.1-10 PM in the medium is required for about 4 min at 20°C in order to trigger the meiosis of 50 % of ripe prophase-blocked Marthasterias glacialis oocytes, and for about 4 min 30 set to trigger the meiosis of virtually 100% of the oocytes (hormone-dependent period) [ 10, 301. Equilibration of 1-MeAde receptors with the hormone is a very rapid process: maximal rate of Ca2+ release, which is expected to be reached only after equilibration of the hormone receptors with the hormone (at least in the classical occupancy theory), is already recorded 0.1 set after 2x lo-’ M addition to plasma membrane-rich fraction (fig. 2). Moreover, the independency of the duration of the hormone-dependent period with hormone concentration from 0.1 PM to 10 PM strongly supports the view that also for intact oocytes, the amount of 1-MeAde bound to the receptors reaches its equilibExp Cell Res 115 (1978)
rium value almost instantaneously as compared with the duration of the hormonedependent period. On the other hand, development of the free Ca2+ peak occurs during the first 10 set following hormone application in intact oocytes and even more rapidly with isolated plasma membranes. Taken together the above results raise the question: why is the interaction of I-MeAde with its plasma membrane receptors required for several minutes (hormone-dependent period) and not only for some seconds? Although the hypothesis cannot be presently ruled out that transduction could be a non linear sequence involving two compulsory and independent triggering processes at the level of the plasma membrane, one of which being the very rapid induction of Ca2+ ions release from the inner part of the plasma membrane, the other one being a hypothetical several minutes requiring process, this hypothesis seems unlikely. Indeed phosphorylation of endoplasmic proteins (a biochemical consequence of hormonal treatment very well correlated with meiosis reinitiation) which stops if l-MeAde is eliminated before the end of the hormone dependent period whereas it continues normally if the hormone is eliminated later on, already proceeds to its maximal rate 2 min after I-MeAde addition. It is more likely that the association of I-MeAde with its receptors induces only a reversible conformational change in the plasma membrane which results in the lowering of the affinity for Ca2+ ions of target proteins localized at the inner part of the plasma membrane. Elimination of the hormone will result in the reassociation of Ca2+ ions with its Ca2+ binding sites if it occurs before trapping by target cortical proteins and by specialized calcium-trapping structures like mitochondria [3 l] and endoplasmic retic-
In vitro release of Ca2+ ulum [32], unless irreversible biochemical modifications occur in the plasma membrane which preclude such a reversal (protein phosphorylations for example). One would expect that slowing down the rate of the trapping or of the biochemical process must increase the duration of the hormonedependent process. We found indeed that temperature decrease from 24 “C to 15 “C increases the duration of the hormone-dependent period from 4 min 30 set to 9 min without modifying the dose-response [33]. Since only Ca2+ ions which have not been already trapped can bind back to the plasma membrane toward an equilibrium state which is intermediate between the initial unstimulated state (high Ca2+ level) and the stimulated final state (low Ca2+ level). We found indeed that membrane potential changes and reaches a steady state value which is intermediate between the unstimulated state and the fully stimulated state if IMeAde is eliminated 1 mm after its application (i.e. after Ca2+ release and before the end of the hormone dependent phase), whereas it does not change and keeps the characteristical value of fully stimulated oocytes if the hormone is eliminated at the end of the hormone-dependent period [26]. That dissociation of the hormone-receptor complex before the end of the hormone dependent period does not allow the plasma membrane to reverse to it initial unstimulated state, but rather fix it in an intermediate state between this initial state and that it had reached at the time of hormone elimination, is also shown by our observation that lo-’ M 1-MeAde application for 2.5 min, which by itself does not trigger meiosis reinitiation, allows a further identical hormone application to be efficient, provided that the two parts of the treatment are not separated by more than 7-8 min at 20°C [30]. Finally it would be expected that
259
very sensitive plasma membrane properties which are already modified by small modification of its Ca2+ content cannot be reversed to any extent, even if dissociation of the hormone-receptor complex occurs very early after Ca2+ ions have been released from the plasma membrane. This is the case for the unmasking of new ouabain-sensitive Na+ pumping sites, which is already fully triggered by application of 10egM I-MeAde 1 min after hormone application: activity of the pump remains at its stimulated level even if the hormone is eliminated immediately after pump stimulation [28]. We hope that this new model will allow a better estimation of the transduction mechanisms which develop in the membrane after hormonal stimulation. We are very grateful to MS C. Guerrier for excellent technical assistance and greatly acknowledge financial support from the DGRST (ACC 77-7-0259).
REFERENCES Kanatani, H, Science 146(1964) 1177. Kanatani, H & Shirai, H, Nature 216 (1%7) 284. Schuetz, A W & Biggers, J D, Exp cell res 46 (1967) 624. Kanatani, H, Exp cell res 57 (1969) 333. - Oogenests (ed J D Biggers & A W Schuetz) p. 459. University Park Press, Baltimore, Md (1972). 6. Kanatani, H, Shirai, H, Nakaniski, K & Kurokawa, T, Nature 221 (1%9) 273. 7. Kanatani, H, Kurokawa, T & Nakaniski, K, Biol bull 137(1%9) 384. 8. Kanatani, H & Hiramoto, H, Exp cell res 61 (1970) 280. 9. Do&e, M & Guerrier, P, Exp cell res 96 (1975) 2%. 10. Do&e, M, Guerrier, P & Leonard, N J, Proc natl acad sci US 73 (1976) 1669. 11. Moreau, M, Guerrier, P & Doree, M, Exp cell res 115 (1978) 245. 12. Moreau, M, Guerrier, P, DorCe, M & Ashley, C C, Nature 272 (1978) 251. 13. Guerrier, P, Moreau, M & Do&e, M, Ann bio) animal biochem biophys 18 ( 1978)441. 14. Guerrier, P, Cahiers de biologie marine (Roscoffl 13 (1972) 475. 1.5. Detering, N K, Decker, G L, Schmell, E D & Lennarz, W J, J cell bio175 (1977) 889. 16. Emmelot, P, Bos, C J, Benedetti, E L & Riimke, P, Biochim biophys acta 90 (1964) 126. Exp Ceil Res II5 (1978)
260
DorPe, Moreau and Guerrier
17. Wilfong, R F & Neville, D M, Jr, J biol them 245 (1970) 6106. 18. Widnell, C C & Unkeless, J C, Proc natl acad sci US 61 (1968) 1050. 19. Wolff, J & Halmi, N S, J biol them 238 (1963) 847. 20. Ahmed, K & Judah, J D, Biochim biophys acta 93 (1964) 603. 21. Emmelot, P & Bos, C J, Biochim biophys acta 150 (1968) 341. 22. Kishimoto, T & Kanatani, H, Exp cell res 82 (1973) 2%. 23. Kishimoto, T, Cayer, M L & Kanatani, H, Exp cell res 101(1976) 104. 24. Guerrier, P, Moreau, M & Do&e, M, Mol cell endocrinol7 (1977) 137. 25. Moreau, M, &terrier, P & DorCe, M, Biol cell 32 (1978) 69. 26. DorCe, M, Guerrier, P & Moreau, M, Actualitts sur les hormones d’invertebrts (ed CNRS) p. 199. CNRS, Paris (1975). 27. Moreau, M & Cheval, J, J physio172 (1976) 293.
28. &terrier, P, Moreau, M & Doree, M. In preparation. 29. Cuatrecasas, P & Hollenberg, M D, Adv in protein chemistry 30 (1976) 251. 30. Guerrier, P& DorCe, M, Dev biol47 (1975) 341. 31. Baker, P F, Calcium in biological systems. SEB symp 30 (1976) 67. 32. Moore, L, Chen, T, Knapp, H R, Jr & Landon, E J, J biol them 250 (1975) 4562. 33. Guerrier, P, Dorte, M, Moreau, M, Cheval, J & Freyssinet, G, Actualitts sur les hormones d’invertebres (ed CNRS) p. 191. CNRS, Paris (1975). 34. Ariens, E J, Arch int pharmacodyn theor 99 (1954) 1, JL. 35. Stephenson, R P, Brit j pharmacol chemother 11 (1956) 379. Received December 23, 1977 Revised version received March 14, 1978 Accepted March 29, 1978
Experimental Cell Research 115 (1978) 251-260
HORMONAL
CONTROL
OF MEIOSIS
In vitro Induced Release of Calcium Ions from the Plasma Membrane in Star-sh Oocytes M. DORRE, M. MOREAU and P. GUERRIER Station Biologique,
F-29211 Roscoff,
France
SUMMARY Using the photoprotein aequorin as a marker, we found that the Ca*+ release, which we demonstrated to occur from the inner part of the plasma membrane after hormonal stimulation of intact starfish oocytes can also be recorded in vitro. Our acellular system was prepared from cortices of Murthasterias glacialis oocytes isolated in Ca*+-free medium and extracted with Triton X-100. At O-PC, the 100000 g pellet obtained from this plasma membrane-rich extract responds in less than 0.1 set to the hormone I-methyladenine, its biological active analogues and mimetics, whereas supematant exhibits a quite lower activity which is no longer present in the head fraction recovered after dialysis through Diatlo UM 2 filters. The in vitro calcium response is competitively inhibited by various agents which, in the same concentration range, act on meiosis reinitiation and free calcium release by the living oocyte. These and previous data are further discussed taking into account the overall chain of events which leads to the release of meiosis inhibition.
In starfish, full grown oocytes are arrested at the prophase stage of meiosis. Meiosis is reinitiated by a hormone produced by the follicle cells [l-5]. The hormone has been identified as I-methyladenine ( I-MeAde) [6-71. It has been demonstrated that lmethyladenine controls meiosis by way of stereospecific receptors localized on the oocyte plasma membrane [8-l 11. Recently, we found that 1-MeAde induces the release of intracellular calcium in Murthasterius glacialis intact oocytes, which is essential for meiosis reinitiation inasmuch as any treatment which suppresses it or decreases it under a threshold value abolishes meiosis reinitiation [12-131. We present here new data showing that this release can be recorded in vitro as an immediate effect of the concentration-dependent and specific interaction of the hormone with a plasma mem-
brane-rich fraction prepared from isolated starfish oocytes cortices.
MATERIAL
AND METHODS
Fractionation procedure glacialis oocytes were prepared free of follicle cells [9] and processed according to the modification of Sakai’s procedure described by &terrier 1141to obtain isolated cortices: oocytes were collected by gentle centrifugation, washed rapidly with NaCl 0.53 M-Tris maleate 0.05 M pH 8.2 (no EDTA was added), which was cleared of traces of calcium through a Chelex 100 column (bed height 30 cm, i.d. 4 cm), and submitted to 10 strokes of a hand homogeniser fitted teflon pestle in ice cold buffer. A pellet was obtained after 1 min centrifugation at 1000 g. The first supematant was collected (endoplasmic fraction). It contained 98% of oocytes proteins and most mitochondria. The 1000 g pellet was washed once again with the same buffer to obtain isolated cortices, which contain the microvilli-rich peripheral envelopes and no characterized mitochondria as shown by electron microscopy. Cortices were resuspended in the same buffer containing 0.2% Triton X-100 and very slowly
Marthasterias
Exp Cell Res 115 (1978)
252
Dorte, Moreau and Guerrier Follicle
cells-free
Potter (buffer
Fractionation of lo5 g supernatant obtained from the plasma membranerich fraction
oocfles
homogeneisation
A 0.53M NaCUL05M passed through a chelex
30 * centrifugatton
Tris-maleate x 100 col”mn
at 1000
pH8.2)
Ultrafiltration of the lo5 g supematant (230 pg protein/ ml) though Diaflo UM2 filters were performed at 4 “C in an Amicon device under 2.1 Atm (30 psi) N2. 500 ~1 dialysates contained 25 pg protein by ml.
g
pjjq+y-q
washed
with
buffer
A
pz+ resuspended for 30 mn at 0°C in 0.2 % Triton in buffer A 5 mn centrifugation
at 1000
g
p(pi/izpJ
Electron microscopy The pellets were fixed in 6% glutaraldehyde, 0.2 M cacodylate buffer, pH 7.4,3.5% NaCI, post-fixed with 1% osmium, 0.2 M cacodylate, 5% NaCl and embedded in Spurr medium. Ultrathin sections were prepared with an LRB ultramicrotome and stained with uranyl acetate and lead citrate.
Handling 40000 10’9
rpm SW 50.145 /\ pellet
In”
10” g supernatant
Fig. 1. Procedure for obtaining the plasma membranerich fraction.
shaken at 0 “C for 30 min before being centrifuged again at 1000 g for 5 min. Aggregated ghosts were recovered in the pellet. The clear supematant, which contained about 40% of the total proteins of the isolated cortices, was collected (“Plasma membrane-rich fraction”). A lo5 g pellet and a 10s g supematant were prepared from this fraction after 45 min centrifugation at 40000 rpm with an SW50.1 rotor in a Beckman L5-75 ultracentrifuge. The protein content was 30-40 fold higher in the supematant than in the pellet. Fig. 1 depicts the flow diagram for preparaing the different subcellular fractions.
of aequorin
Aequorin, grade I, purchased from Sigma, was puritied by chromatography through small G25 Sephadex columns equilibrated with 1 pm M EDTA., 10 mM sodium acetate, pH 6.2. 50 /~1aequorin solutions were put in small plastic preparation dishes mounted directly on the photomultiplier enclosed in a light-tight cage. Subcellular fractions (500 J) and chemicals (50 ~1) were injected into the aequorin solution by remote control through a plastic syringe. Flash emissions were recorded as already described [12]. From 0.05 to 0.4 nA, increases of anode current were found to be proportional to the quantities of Ca*+ introduced or released in the preparation dish. Although we always worked in this range, we found it better to express our results in arbitrary units since calibrations performed as indicated on fig. 3A showed that the light response (anode current deviation) recorded after adding a known amount of Ca*+ to the vessel may vary from one batch of aequorin to the other.
RESULTS Enzymes assays The reaction mixture for 5’-nucleotidase assay was made from 50 ~1 &Y-AMP low3 M (0.1 &i), 100 ~1 Tris lo-’ M, pH 8.5, MgCI, lo-* M and 50 ~1 sub-cellular fraction. 50 ~1 aliquots were collected as a function of time, boiled for 3 min after injection into 150~1 distilled water, and passed through a small Pasteur pipette filled with 1 ml DEAE Sephadex. The pipette was rinsed with 2 ml of distilled water and the eluate containing adenosine (and eventually adenine formed by nucleoside hydrolase interfering activity) as the reaction product, was counted for radioactivity. The reaction mixture for alkaline nitrophenylphosphatase was made from 200 ~1 paranitrophenyl-phosphate lo-* M, 200 ~1 Tris 0.2 M MgCI,, 5 mM, pH 9, and 200 ~1 subcellular fraction. Release of nitrophenol was measured at 420 nm. Subcellular fractions for determination of enzymic activities contained 0.5-2 mg proteins/ml. Exp Cell Res 115 (1978)
Fig. 2A shows a phase contrast micrography of cortices isolated from Murthasterius glacialis oocytes. Such isolated cortices include vitelline membrane, plasma membrane and the 2-5 pm superficial layer of the oocyte . This cortical ghost first fraction which was found to possess adenylate cyclase and phosphodiesterase activity (cryptic and not cryptic), readily responded to hormonal stimulation by releasing free calcium in the medium. Controls were run using aequorin + ghosts or aequorin + I-MeAde prepared
In vitro release of Ca2+
Fig. 2. (A) Isolated cortical ghosts obtained by homogenization in the extraction medium. The bar represents 20 PM. (B) Electron micrograph of the 1oJ g pellet fraction obtained from the low speed supematant after extracting the cortical ghosts 30 min at 0 “C with 0.2% Triton X-100. The bar represents 0.5 pm.
in the extraction medium which always gave negative results. Mild treatment with diluted detergents releases a fraction containing a homogeneous population of membrane vesicles ranging from 0.1 to 0.4 pm in diameter which is re-
253
covered in the 105g pellet (fig. 2B). Some sections demonstrated, however, the presence of scarce contaminating electron dense and fibrous material which is likely to derive from cortical granules which may have been destroyed during preparation in our EDTA- and EGTA-free medium [IS]. Significant S-nucleotidase and alkaline nitrophenylphosphatase activities were found to be associated with the membrane vesicles (table 1). Since the enzymes have been demonstrated to be specific plasma membrane markers [ 16-211 the fraction containing the membrane vesicles was referred as the “plasma membrane-rich fraction” in this text, although most of its proteins remains in the lo5 g supernatant after centrifugation. Fig. 3B shows an original record of the response displayed on the oscilloscope when 2x lo-’ M I-MeAde was added to the plasma membrane-rich fraction in a typical experiment. Less than 0.1 set after hormone addition, the anode current increased from 0 to 0.5 nA, which corresponds to an increase of free Ca2+ions in the vessel from 0 to about 2x lo-l2 ion g (fig. 3A). Ten seconds after hormone addition, free Ca2+level had already come back nearly to zero. Table 2 shows that only those analogues of 1-MeAde which can replace the hormone in triggering meiosis reinitiation of intact
Table 1. Specific activities of two plasma membrane marker enzymes in subcellular fractions from Marthasterias glacialis oocytes a Isolated cortices Plasma membrane rich fraction Enzymes
Endoplasm
Cortical pellet
Total
105g pellet
l@g supematant
5’ nucleotidase Alkaline nitrophenylphosphatase
2 0.1
35.2 0.75
2 0.1
44 0.9
1 0.02
a Expressed as pmoles substrate transformed/h/mg proteins. Exp Cell Res I I5 ( 1978)
254
Dorke, Moreau and Guerrier
Fig. .?. Light responses recorded on the oscilloscope. (A) Calibration: lo-‘* ion g Caz+ (0.05 ml of a 20 nM
Ca*+ solution) were added to 0.4 ml of the extraction medium + 0.03 ml of aequorin solution. Identical value was recorded when extraction medium was substituted by the plasma membrane-rich fraction used in B and C, which reoresented extracted cortices from about 2X lo5 oocytes; (8) response following stimulation with 2X lOA7M I-MeAde (final concentration): (C) response obtained with 10 mM DTT (final cdncentration). Vertical and horizontal bars correspond respectively to 0.12 nA anode current and to 12 sec.
oocytes are able to release Ca*+ ions from the plasma membrane-rich fraction. Moreover, fig. 3C shows that addition of dithiothreitol (DTT), which mimics I-MeAde in inducing meiosis reinitiation [22], increasing protein-SH in the oocyte cortex [23] and stimulating protein phosphorylation [24] also results in an important release of Ca2+, although the quite different time course of CaZf release suggests that DTT does not act as specifically as the hormone and releases also Caz+ from membranes sites which are not essential for meiosis reinitiation. Fig. 4 shows that 1-MeAde triggers only a very small release of Ca2+ ions from the endoplasmic fraction, in contrast with the two fractions obtained from isolated cortices. Moreover, it makes clear that Ca2+ release is higher from the plasma membrane-rich fraction than from the residual cortical pellet. The lo5 g supernatant obtained from the plasma membrane-rich fraction, which contains most proteins, only slightly releases Ca2+ions following lMeAde addition, in contrast with the lo5 g pellet, which contains the plasma memExp Cell Res I15 (1978)
brane vesicles. No activity is found in the head fraction recovered after dialysis on Diaflo UM2 filters. Fig. 5A shows the response of the same batch of plasma membrane-rich fraction to stepwise addition of I-MeAde. Ca*+ release is already noticeable with 2~ low9 M l-MeAde and stepwise increase of the 1-MeAde concentration from 2x lo+ M to 2x 10e6M enhances the amount of Caz+ ions released after each hormone addition. However, repeated hormone additions at high concentration exhaust the ability of the plasma membrane-rich fraction to release further Ca2+ions, although less than 5 % of the total membrane-bound Cal+ has been previously released (as shown after injuring the plasma membrane-rich fraction with 20 % Triton X100). We have shown that methylxanthines competitively inhibit meiosis reinitiation and stimulation of protein phosphorylation induced by both 1-MeAde and DTT [lo, 241. Fig. SA shows that theophyllin (Sigma) also competitively inhibits the hormone-induced Ca2+ release from the plasma mem-
Table 2. Ability of various structural analoguesa to replace I-MeAde in triggering meiosis reinitiation and calcium release from plasma membrane-rich fraction Compounds 1-Methyladenine I-Ethyladenine 1Benzyladenine I-Isopentenyladenine 1,N, 6Dibenzyladenine
Meiosis reinitiation
Calcium release
Active
Active
Inactive
Inactive
I
~:$:“,:~~~P~~~nthine I-Methyladenosine
1~o$r$o$p~a$e-5’1,7-Dimethyladenine
I
n All compounds were used at the concentration to-6M.
In vitro release of Cazf
A
B
255
C
Fig. 4. Abscissa: subcellular fractions tested for Ca*+ release after addition of 10-r M I-MeAde; ordinate: amount of Cal+ ions released per 138 pg of proteins (arbitrary units proportionally to anode current). Isolated cortices were fractionated into a plasma membrane-rich fraction (A) and a residual cortical pellet (B) according to the procedure described in fig. 1. Fraction A and endoplasm (C) were tested for Cal+ release before (Z’) or after subfractionation into a 105g pellet (P) and the corresponding supematant (S). lMeAde 10e6M in 0.05 ml was added to a 0.5 ml assay containing respectively 102 pg (A, S); 92 ,ug (A, P); 138 pg (A, T); 120 pg (B) and 70 pg proteins (C, 7’). Original recorded values for these concentrations appear in black, hatched area represents adjustment up to 138pg proteins.
brane-rich fraction, Essentially identical results were obtained with caffeine (Sigma) or when DTT was substituted for I-MeAde. Similar inhibitions of 1-MeAde induced Ca2+ release from plasma membrane-rich fractions were recorded with various agents which block meiosis reinitiation and free calcium release in the living oocyte [12, 241 such as 10 mM MnCl,, 0.5 mM D 600 (Knoll), 5 mM procaine hydrochloride (Merk) or N-ethyl maleimide 0.1 mM (Sigma) and Emetine 400 pg/ml (Sigma). In all cases inhibition of Ca2+ release was released by raising 1-MeAde concentration. Competitive inhibition of the calcium and biological responses occurred in the same range of concentrations, both in vivo and in vitro [25]. Plasma membrane-rich fractions were now prepared from oocytes which had been
Fig. 5. Abscissa: I-MeAde concentrations; ordinate: amounts of Ca*+ ions released per mg of proteins (in arbitrary units proportionally to anode currents). Effect of stepwise increases of I-MeAde concentration on the amount of Cazf ions released from the plasma membrane-rich fraction. (A) A plasma membrane-rich fraction prepared from prophase-blocked oocytes (before hormonal treatment) was divided into two batches, one of which received theophyllin to the final concentration 5x 10-s M, the other serving as control. I-MeAde was added stepwise to the final concentration indicated (from 2x lO-9 M to 4x lOA M for the control; from 2x 10m9M to 2x lOmEM for the theophyllin-treated batch. The height of each block refers to the amount of CaZ+ ions released in the control batch, the height of the black area to the amount of CaZ+ released in the theophyllin treated batch. (B) Plasma membrane-rich fraction prepared from oocytes which had been first treated for 8 min with 2x 10-OM I-MeAde: stepwise addition of I-MeAde to the final cont. 2X 10-r M. 5x 10-r M and low6M.
already treated for 8 min with 2X lo-” M lMeAde and then washed free of hormone. Fig. 5B shows that addition of 2~ lo-’ M 1-MeAde elicits only a very small residual release of Ca2+ ions, which cannot be increased by further hormone application.
DISCUSSION The present results show that a specific release of free Ca2+ions from plasma membrane-rich vesicles obtained from isolated cortices is triggered by 1-MeAde, its active analogues and its mimetic agent, DTT. It is very likely that the hormone cannot release Ca2+ ions from mitochondria or endoplasmic reticulum which constitute the main Exp CdRes
115 (1978)
256
0’
Dot-de, Moreau and Guerrier
’ 10-3
I 10-z
I 10-l
, 1
1
I
10
100
I
Fig. 6. Abscissa: Hormone concentration in micromoles; ordinate: receptors occupancy. Schematic representation of receptor occupancy as derived from the amount of Ca*+ released in intact oocytes treated with I-MeAde. The curve represents the quantities of light emitted by aequorin-injected oocytes following addition of I-MeAde at various concentrations [12]. Levels of receptors occupancy for maximal values of the various elementary responses are shown. a, Threshold for in vivo and in vitro calcium release; b, saturation for Na+, K+-dependant ATPase; c, saturation for stimulation of protein phosphorylation and for meiosis reinitiation.
membrane systems of the endoplasmic fraction, since the very small Ca2+ release found with this fraction could be accounted for by the contamination by some peripheric membrane fragments which would be expected. Since removal of the vitelline membrane neither reduces the calcium release induced by the hormone in intact oocytes [12] nor modifies the biological responses [26, 271, the present results strongly support the view that only the plasma membrane releases Ca2+following hormone application. This agrees very well with our previous studies which demonstrated the absence of intracellular receptors for lMeAde recognition [ 1l] and established that I-MeAde receptors are exclusively localized at the level of the plasma membranes [8, 91. In this study, we found that a plasma membrane-rich fraction extracted Exp Cell Res 115 (1978)
from about 2 x lo5 oocytes released 2 x lo-l2 Ca2+ ion g. This corresponds to a basal release of about lo-l7 ions by oocyte cortex, which would account for a 5 nM variation if adjusted to the 1760picoliters oocyte volume. When compared with the micromolar range variation found in vivo [12, 131, this points to a 1% recovery, following our procedure. Since higher values were also obtained in other experiments (up to 10F” ion g), it seems likely that Ca2+ binding sites are more readily accessible to hormone stimulation in the in vitro than in the in vivo conditions. Another possibility may be worth considering, however: that our preparation has been complemented with membranes originating from cortical granules lysed during triton extraction and which may eventually respond to hormonal stimulation. This makes little difference, however, since one can not argue that our system has become by the fact highly different from the membrane 1-MeAde interacts with normally. We found indeed that germinal vesicle-bearing oocytes, which were preactivated by ionophore A 23 187 and have released cortical granules matrix [13], readily responded to the hormone and were even more sensitive than untreated ones. In these conditions, 50% meiosis was reached, using 6x lo+? M 1-MeAde, instead of the 1.4~ 10e7 M concentration required with untreated controls from the same batch. Ca2+release from the plasma membrane, stimulation of the ouabain sensitive Na+/K+ pump [28], stimulation of protein phosphorylation [24] and meiosis reinitiation [lo] share in common the same specificity. The only active structural analogs of 1-MeAde are Nl-substituted adenines; activity is conserved or even increased when the methyl group is replaced by larger substituent groups; activity is lost when N l-substituted
In vitro release of Ca2’
257
adenines are further substituted at the N7 be considered for such a process: these may or at the N9 position, whereas it is con- be a direct displacement by the hormone of served in Nl, N6 disubstituted adenines. Ca2+ normally bound to the receptor site. This strongly supports the view that all Alternatively, the effect of the hormone these biological effects are consequences of may be considered as an allosteric linkage I-MeAde interaction with the same cate- with the hormone serving (when bound to gory of plasma membrane receptors. Ob- its specific recognition site) to reduce the tention of similar concentration dependen- affinity of the Ca2+ binding site. This latter cies for binding and biological response has mechanism, which assumes two different often been considered as evidence that the sites on the plasma membrane for both redetected binding sites were the receptors in- cognition function and effector function volved in the biological response, although (calcium release) seems to be more plausithe molecular mechanism involved in the ble since previous studies based on microtransduction was unknown. However, our injection techniques [8, 111 or competitive system clearly shows that the threshold inhibition of labelled I-MeAde uptake [9] value of hormone concentration required to have established that the hormone receptor obtain a biological effect may depend on the is accessible to 1-MeAde from the outside nature of the biological effect under inves- of the cell and not from the inside, whereas tigation: maximal stimulation of the Na+/K+ Ca2+ release occurs exclusively on the inpump is already obtained with nanomolar side of the plasma membrane [12]. The rehormones concentrations [28], maximal ceptor and effector function may reside at stimulation of protein phosphorylation and two different sites of the same molecule, or meiosis reinitiation occur in the 0.1-0.2 PM more likely, reside in separate molecules range [lo, 241 whereas maximal Ca2+ re- which interact directly or indirectly in the lease is reached only with micromolar hor- fluid mosaic membrane. We showed previmone concentrations. According to the oc- ously that Ca2+ release already occurs l-2 cupany theory [34, 351, this would imply set after application of 1-MeAde to intact that a maximal effect at the level of the oocytes [12, 131;now we found that isolated Na+/K+ pump is achieved with only a mi- plasma membranes already release Ca2+ nority of receptors occupied. The additional ions less than 0.1 set after 2~ lo-’ M l“spare” receptors [29] are not inactive re- MeAde addition at 4°C. On the other hand ceptors since the other biological responses suppression of calcium release by methylrequire greater level of receptors occupan- xanthines, MnCl,, D600 or procaine supcy. Even meiosis reinitiation, however, presses both metabolic activation [24, 281 which is the most integrated biological re- and meiosis reinitiation [lo, 12, 131. These sponse to the hormone, does not require full findings strongly support the view that Ca2+ occupancy of the receptors. Fig. 6 gives a release from the plasma membrane into the schematic picture of receptors occupancy cortical region of the oocyte is the first step for the various responses of the oocytes to in the sequence of events leading to metabolic activation and meiosis reinitiation. It hormone application. Inasmuch as both interaction of 1-MeAde is very likely that loss of Ca2+ from the with its stereospecific receptor and release plasma membrane is not by itself sufficient of Ca2+ions occur at the level of the plasma to initiate this sequence and that the free membrane, two possible mechanisms may Ca2+ concentration must actually increase Exp Cell Res I IS f 1978)
258
Dot-&e. Moreau and Guerrier
transiently (probably only in the oocyte cortex) since EGTA, which would not be expected to inhibit Ca2+ ions release, suppresses meiosis reinitiation if it is injected into intact oocytes before development of the free Ca2+peak, i.e. less than 10 set after hormone addition [ 121. Protein phosphorylation is stimulated more rapidly (less than 2 min after hormone addition) and extensively in the cortex of the oocyte than in the endoplasm, suggesting that phosphorylation of some cortical proteins might be a major event in meiosis reinitiation [24]. This agrees with our results which suggest that the target for Ca2+-induced metabolic activation must be located in the vicinity of the plasma membrane, although the exact mechanism still remains to be elucidated. The finding by Kishimoto & Kanatani that the protein-SH content increases in the cortex following hormone addition points in the same direction [22]. We showed elsewhere that the presence of I-MeAde in the range 0.1-10 PM in the medium is required for about 4 min at 20°C in order to trigger the meiosis of 50 % of ripe prophase-blocked Marthasterias glacialis oocytes, and for about 4 min 30 set to trigger the meiosis of virtually 100% of the oocytes (hormone-dependent period) [ 10, 301. Equilibration of 1-MeAde receptors with the hormone is a very rapid process: maximal rate of Ca2+ release, which is expected to be reached only after equilibration of the hormone receptors with the hormone (at least in the classical occupancy theory), is already recorded 0.1 set after 2x lo-’ M addition to plasma membrane-rich fraction (fig. 2). Moreover, the independency of the duration of the hormone-dependent period with hormone concentration from 0.1 PM to 10 PM strongly supports the view that also for intact oocytes, the amount of 1-MeAde bound to the receptors reaches its equilibExp Cell Res 115 (1978)
rium value almost instantaneously as compared with the duration of the hormonedependent period. On the other hand, development of the free Ca2+ peak occurs during the first 10 set following hormone application in intact oocytes and even more rapidly with isolated plasma membranes. Taken together the above results raise the question: why is the interaction of I-MeAde with its plasma membrane receptors required for several minutes (hormone-dependent period) and not only for some seconds? Although the hypothesis cannot be presently ruled out that transduction could be a non linear sequence involving two compulsory and independent triggering processes at the level of the plasma membrane, one of which being the very rapid induction of Ca2+ ions release from the inner part of the plasma membrane, the other one being a hypothetical several minutes requiring process, this hypothesis seems unlikely. Indeed phosphorylation of endoplasmic proteins (a biochemical consequence of hormonal treatment very well correlated with meiosis reinitiation) which stops if l-MeAde is eliminated before the end of the hormone dependent period whereas it continues normally if the hormone is eliminated later on, already proceeds to its maximal rate 2 min after I-MeAde addition. It is more likely that the association of I-MeAde with its receptors induces only a reversible conformational change in the plasma membrane which results in the lowering of the affinity for Ca2+ ions of target proteins localized at the inner part of the plasma membrane. Elimination of the hormone will result in the reassociation of Ca2+ ions with its Ca2+ binding sites if it occurs before trapping by target cortical proteins and by specialized calcium-trapping structures like mitochondria [3 l] and endoplasmic retic-
In vitro release of Ca2+ ulum [32], unless irreversible biochemical modifications occur in the plasma membrane which preclude such a reversal (protein phosphorylations for example). One would expect that slowing down the rate of the trapping or of the biochemical process must increase the duration of the hormonedependent process. We found indeed that temperature decrease from 24 “C to 15 “C increases the duration of the hormone-dependent period from 4 min 30 set to 9 min without modifying the dose-response [33]. Since only Ca2+ ions which have not been already trapped can bind back to the plasma membrane toward an equilibrium state which is intermediate between the initial unstimulated state (high Ca2+ level) and the stimulated final state (low Ca2+ level). We found indeed that membrane potential changes and reaches a steady state value which is intermediate between the unstimulated state and the fully stimulated state if IMeAde is eliminated 1 mm after its application (i.e. after Ca2+ release and before the end of the hormone dependent phase), whereas it does not change and keeps the characteristical value of fully stimulated oocytes if the hormone is eliminated at the end of the hormone-dependent period [26]. That dissociation of the hormone-receptor complex before the end of the hormone dependent period does not allow the plasma membrane to reverse to it initial unstimulated state, but rather fix it in an intermediate state between this initial state and that it had reached at the time of hormone elimination, is also shown by our observation that lo-’ M 1-MeAde application for 2.5 min, which by itself does not trigger meiosis reinitiation, allows a further identical hormone application to be efficient, provided that the two parts of the treatment are not separated by more than 7-8 min at 20°C [30]. Finally it would be expected that
259
very sensitive plasma membrane properties which are already modified by small modification of its Ca2+ content cannot be reversed to any extent, even if dissociation of the hormone-receptor complex occurs very early after Ca2+ ions have been released from the plasma membrane. This is the case for the unmasking of new ouabain-sensitive Na+ pumping sites, which is already fully triggered by application of 10egM I-MeAde 1 min after hormone application: activity of the pump remains at its stimulated level even if the hormone is eliminated immediately after pump stimulation [28]. We hope that this new model will allow a better estimation of the transduction mechanisms which develop in the membrane after hormonal stimulation. We are very grateful to MS C. Guerrier for excellent technical assistance and greatly acknowledge financial support from the DGRST (ACC 77-7-0259).
REFERENCES Kanatani, H, Science 146(1964) 1177. Kanatani, H & Shirai, H, Nature 216 (1%7) 284. Schuetz, A W & Biggers, J D, Exp cell res 46 (1967) 624. Kanatani, H, Exp cell res 57 (1969) 333. - Oogenests (ed J D Biggers & A W Schuetz) p. 459. University Park Press, Baltimore, Md (1972). 6. Kanatani, H, Shirai, H, Nakaniski, K & Kurokawa, T, Nature 221 (1%9) 273. 7. Kanatani, H, Kurokawa, T & Nakaniski, K, Biol bull 137(1%9) 384. 8. Kanatani, H & Hiramoto, H, Exp cell res 61 (1970) 280. 9. Do&e, M & Guerrier, P, Exp cell res 96 (1975) 2%. 10. Do&e, M, Guerrier, P & Leonard, N J, Proc natl acad sci US 73 (1976) 1669. 11. Moreau, M, Guerrier, P & Doree, M, Exp cell res 115 (1978) 245. 12. Moreau, M, Guerrier, P, DorCe, M & Ashley, C C, Nature 272 (1978) 251. 13. Guerrier, P, Moreau, M & Do&e, M, Ann bio) animal biochem biophys 18 ( 1978)441. 14. Guerrier, P, Cahiers de biologie marine (Roscoffl 13 (1972) 475. 1.5. Detering, N K, Decker, G L, Schmell, E D & Lennarz, W J, J cell bio175 (1977) 889. 16. Emmelot, P, Bos, C J, Benedetti, E L & Riimke, P, Biochim biophys acta 90 (1964) 126. Exp Ceil Res II5 (1978)
260
DorPe, Moreau and Guerrier
17. Wilfong, R F & Neville, D M, Jr, J biol them 245 (1970) 6106. 18. Widnell, C C & Unkeless, J C, Proc natl acad sci US 61 (1968) 1050. 19. Wolff, J & Halmi, N S, J biol them 238 (1963) 847. 20. Ahmed, K & Judah, J D, Biochim biophys acta 93 (1964) 603. 21. Emmelot, P & Bos, C J, Biochim biophys acta 150 (1968) 341. 22. Kishimoto, T & Kanatani, H, Exp cell res 82 (1973) 2%. 23. Kishimoto, T, Cayer, M L & Kanatani, H, Exp cell res 101(1976) 104. 24. Guerrier, P, Moreau, M & Do&e, M, Mol cell endocrinol7 (1977) 137. 25. Moreau, M, &terrier, P & DorCe, M, Biol cell 32 (1978) 69. 26. DorCe, M, Guerrier, P & Moreau, M, Actualitts sur les hormones d’invertebrts (ed CNRS) p. 199. CNRS, Paris (1975). 27. Moreau, M & Cheval, J, J physio172 (1976) 293.
28. &terrier, P, Moreau, M & Doree, M. In preparation. 29. Cuatrecasas, P & Hollenberg, M D, Adv in protein chemistry 30 (1976) 251. 30. Guerrier, P& DorCe, M, Dev biol47 (1975) 341. 31. Baker, P F, Calcium in biological systems. SEB symp 30 (1976) 67. 32. Moore, L, Chen, T, Knapp, H R, Jr & Landon, E J, J biol them 250 (1975) 4562. 33. Guerrier, P, Dorte, M, Moreau, M, Cheval, J & Freyssinet, G, Actualitts sur les hormones d’invertebres (ed CNRS) p. 191. CNRS, Paris (1975). 34. Ariens, E J, Arch int pharmacodyn theor 99 (1954) 1, JL. 35. Stephenson, R P, Brit j pharmacol chemother 11 (1956) 379. Received December 23, 1977 Revised version received March 14, 1978 Accepted March 29, 1978