THE JOURNAL OF EXPERIMENTAL ZOOLOGY 24623-32 (1988)

Conditions for the Appearance of Maturation Promoting ~

Factor Following Germinal Vesicle Disruption of Prophase-Arrested Starfish Oocytes

P. GUERRIER, I. NEANT, M. CHARBONNEAU, AND M. MOREAU Station Bwlogique, 29211 Roscoff(l? G., IN. , M.M.) and Facult.6 des Sciences, 35042 Rennes, France (M. C.)

ABSTRACT In this paper, we report that so-called "incompetent" prophase-arrested oocytes of Marthasterias glacialis and Asterias rubens fail to produce any aster or spindle when their nucleo- plasm and cytoplasm are mixed via pressure or microinjection. We also show that the same incompe- tent oocytes always complete their maturation up to second polar body extrusion and female pronucleus formation following a 30 sec shaking treatment, which results at once in germinal vesicle (GV) disruption and produces the same mixing of their nuclear and cytoplasmic compartments.

32P phosphate incorporation into proteins and cell fusion experiments indicates that this mechani- cal treatment induces an increased protein phosphorylation and triggers the formation of active maturation promoting factor (MPF) molecules which are not produced following a simple nucleoplasm transfer. While bovine serum albumin (BSA) had no effect on this process, the intracellular Ca2' antagonists (2-[(2-bis[Carboxymethyl~-amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis~carboxy- methyl~-aminoquinolinetetrakis-[acetoxymethyl]ester) (Quin 2-AM) and 8(N-N-diethylamino) octyl- 3,4,5-trimethoxybenzoate (TMB 8) were found to inhibit both hormone-induced and shaking-induced maturations. These results exclude the possibility that shaking might act by releasing arachidonic acid in the external medium. Instead, they suggest that such a mechanical treatment may trigger the same significant changes in Ca2+ concentration or compartmentalization that have been shown to occur following hormone stimulation and that seem to be required for activating resting female centrioles as well as latent MPF precursors and protein kinases.

Full grown starfish oocytes are arrested at the germinal vesicle (GV) stage during prophase of the first meiotic division. They resume meiosis when exposed to the natural hormone l-methyladenine (l-MeAde), which is produced by the follicle cells under the influence of a small peptide of neural origin (Kanatani and Shirai, '67; Kanatani et al., '69; Kanatani, '73; Schuetz and Biggers, '67). Upon stimulation by the hormone,the oocytes undergo many morphological, biochemical, and physiologi- cal changes (reviewed in Meijer and Guerrier, '84). Thus, l-MeAde has been shown to act on specific recognition sites located on the plasma membrane (Kanatani and Hiramoto, '70; Doree and Guerrier, '75; Doree et al., '76; Moreau et al., '71) to release intracellular bound calcium within 30 sec (Doree et al., '78; Moreau et al., '78; Guerrier et al., '78; '79; Moreau and Guerrier, '79, '80) and stimulate 20-fold the rate of endogenous protein phosphory- lation as early as 2 min after its addition (Guerrier et al., '77a). It acts at the posttranslational level (Guerrier and Doree, '75) to produce maturation promoting factor (MPF), the universal factor re- sponsible for G2-M phase transition, which trig- gers germinal vesicle breakdown (GVBD) and

@ 1988 ALAN R. LISS, INC.

maturation divisions when microinjected into im- mature GV-blocked oocytes (Masui and Markert, '71; Kishimoto and Kanatani, '76; Gerhart et al., '84). Besides the hormone l-MeAde, various non- hormonal stimuli such as dithiothreitol (Kishimoto et al., '76; Guerrier et al., '77a), increased extracel- lular calcium (Guerrier et al., '78), arachidonic acid (Meijer et al., '84), methylglyoxal-bis-guanylhydra- zone (Meijer and Guerrier, ,831, shaking (Guerrier et al., ,831, or nucleoplasm microinjection (Picard and Doree, '84) have also been found capable of inducing meiosis reinitiation, at least in some species.

After hormonal stimulation, starfish oocyte MPF was found to appear earlier in the nucleus than in the cytoplasm and, in Marthasterias glacialis, a related factor has even been shown to be present in the nucleoplasm of some unstimulated GV-ar- rested oocytes. Indeed, while GV-blocked oocytes of Asterias rubens and most starfishes (Kishimoto et al., '81) never respond to the injection of their own nucleoplasm or the nucleoplasm taken from an- other unstimulated oocyte, two classes of recipient

P. Guerrier's present address is Ecole Normale Superieure, 69007 Lyon, France

P. GUERRIER ET AL. 24

oocytes (competent and incompetent) have been de- MeAde to induce maturation or submitted to non- scribed to occur in Marthasterim glacialis. While hormonal stimuli. Increased external calcium con- competent oocytes were found to produce an active centrations were obtained by mixing various MPF within 20 min and to complete maturation amounts of artificial sea water (SW) or calcium up to the female pronucleus stage, incompetent free sea water (CFSW) [prepmed according to oocytes could not form MPF (Picard and Doree, Shapiro ('401 with a 0.32 M isoosmotic solution of '84). These data contrast to previous observations CaClz (Guerrier et al., '78). Shaking was performed which showed that GV-disruption and further mat- for 15 to 30 sec in SW or CFSW, using 3 ml aliquots uration could be mechanically induced by shaking of an oocyte suspension placed in 10 ml test tubes vigorously any oocyte suspension of either Asterias covered with parafilm (Guerrier et al., '83). Since rubens or Marthasterim glacialis (Guerrier et al., the oocytes tend to stick together after this treat- '83). Indeed, when prepared in that way, all of ment, they were further treated for 20 min with these oocytes, including those from Asterim ru- 0.05% pronase in order to digest all the extracel- hens, were found to behave as usually described Mar material. Oocytes were then returned either for Marthasterim glacialis competent oocytes. They to SW or CFSW. completed maturation up to second polar body ex- trusion and female pronucleus formation, acquired Test of oocyte competence full cytoplasmic maturity, and developed normally Mixing of GV-nucleoplasm and cytoplasm was after monospermic fertilization. obtained according to two different procedures

The experiments described in this paper have which involved either externally applied pressure been carried out in an attempt to understand why or microinjection. Microinjection was performed shaking-which produces the same mixing of GV according to the method of Hiramoto ('74), as pre- nucleoplasm and cytoplasm that is obtained by cisely described in the reviews of Meijer et al. ('84) miCrOinJeCtiOn-iS more efficient in triggering full and Kishimoto ('86). A micropipet was inserted maturation. We thus compared the effects of both into the oocyte from the vegetal pole and nucleo- treatments on a number of different batches of plasm was sucked up and immediately reinjected oocytes that were recovered during the breeding into the Same or another oocyte. At least 10 mi- season. SimultaneouslY, we also checked if ComPe- croinjections were performed for each batch tested. tent and incompetent oocytes might differ in their Alternatively, oocytes mounted between slide and potential response to increased or decreased levels CoversIip were compressed by removing excess fluid of extracelhlar and intracehlar Ca2+. Finally, we at the edge of the preparation in order to produce used Polyethylene glYCO1 (PEGI-induced cell fusion GV envelope disruption. In both cases, competent to check for the Presence of MPF in the cytoplasm oocytes were found to develop female asters within of oocytes which were either stimulated by hor- 10 min, while incompetent oocytes did not form mone, shaking, or by increasing extracellular Ca2+ these structures and exhibited a n intact nucleolus concentration. for many hours. They did not differ, in other re-

ow data show that the shaking Procedure trig- spects, from competent oocytes. They showed the Wrs more than a simple mixing ofthe nucleoplasm same degree of chromosome condensation (tetrads) and cytoplasm since it also activates those MPF as observed with Hoechst bisbenzimide H33342 precursor molecules which remain unaffected fol- fluorochrome. Moreover, such GV disrupted incom- lowing a simple nucleoplasm transfer. The fact petent oocytes were always found to complete mat- that in t racehlar Ca2+ antagonists block the uration following hormone addition. meiosis-promoting effect of mechanical stimula- tion and inhibit 1-MeAde-induced maturation sug- gests, moreover, that shifts in intracellular Ca2+ PEG-induced cell fusion must play a significant role in initiating both MPF This very sensitive technique, which transfers precursor activation and the mobilization of the 100% of the cell volume (Vassetsky et al., '83; Bulet meiotic female asters which, usually occur only et al., '85; Guerrier and Nbant, '86), has been se- after hormone-induced nuclear membrane dis- lected to check for the presence of MPF in the rup tion. cytoplasm of oocytes that were either submitted to

MATERIALS AND METHODS shaking or to an increased external Ca2+ concen- tration. In our modified procedure, vitelline enve-

Immature starfish oocytes were prepared free of lope-free oocytes, both unkimulated and those that follicular cells as described previously (Doree and had been previously submitted to nonhormonal Guerrier, '75). They were either treated with I- stimuli up to GVBD, were placed together in a

EVOCATION OF MPF 25

petri dish containing CFSW. Then, a drop of a neously shaken for 30 sec and treated for 20 min viscous solution of PEG and CFSW (l:l, w/w) was more in the presence of the drugs and 0.05% pro- laid down in the center of the preparation and the nase to remove the vitelline layer. The oocytes oocytes gently swirled to its top for 2-4 min. Het- were finally suspended in 10 vol SW or CFSW. erologous pairs, consisting of a GV-arrested oocyte Blocked oocytes were released from their inhibi- or a n ootid and a stimulated oocyte undergoing tion by adding increasing concentrations of 1- maturation, were easily recognized under the dis- MeAde. Dose-response curves were also drawn us- secting microscope. They could not be confused ing oocytes that had been pretreated only with the with pairs including a spontaneously maturing drugs for 30 min, washed, and further stimulated partner (about 5% of the original oocyte popula- with the hormone. Controls from the same batches tion), since fusions were always performed more received the hormone in the presence of the same than 3 h after preparing the oocytes, at which time concentration of the drug vehicle. spontaneously responding oocytes already pre-

Cytological obseruations sented a well developed female pronucleus. Exper- imental pairs were immediately collected and Events like GVBD, aster and spindle formation, transferred to recipient bowls. Further develop- polar body extrusion, and female pronucleus for- ment was observed immediately or after the pairs mation were easily observed in vivo (Fig. 1). More had been mounted on slides in a drop of CFSW detailed observations were performed with an that was protected by a ring of paraffin oil disposed Olympus BH-2 microscope mounted for epifluo- between two fragments of thin glass that SUP- rescence. Thus, chromosome condensation was fol- ported the coverslip. lowed in vivo after staining the oocytes with 1 pM

of Hoechst bisbenzimide H 33342. Localization of Incorporation of phosphate into protein acidic granules in the oocyte cortex was checked

Prophase-arrested oocytes were preloaded with using the fluorescent amine 9-aminoacridine. The 10 pCi/ml 32P carrier-free orthophosphate (Amer- presence of two resting female meiotic centers in sham, 40 pCi/mM) for 4 h and washed until free of close contact with the intact GV, as well as spindle external label. 1-ml aliquot samples containing formation were observed after tubulin immuno- about 6 x lo3 oocytes were injected into 4 ml ice- staining. Briefly, a drop of prophase-arrested oo- cold 10% trichloroacetic acid (TCA), washed twice cytes was extracted for 20 rnin to 1 h in a micro- in the same medium, dissolved in 1 ml 0.5 N so- tubule stabilizing medium containing 1% Triton dium hydroxide (NaOH), and reprecipitated over- XlOO (Paweletz et al., '84). The extracted oocytes, night. The final pellet was washed with 4 ml TCA which were stuck upon a slide covered with a film and dissolved in 1 m10.5 N NaOH. Protein concen- of glycerinated egg albumin, were fixed for at least tration was determined on triplicate aliquots 20 min at room temperature in ethanol-acetic (Lowry et al., '51) and counts per minute recorded acid-distilled water (95:2:3), hydrated, and per- from triplicate 0.5 ml samples suspended in 8 ml meabilized for 30-45 min in Sorensen phosphate liquid scintillant (ACS, Amersham). buffer containing 1% BSA and 0.03% Saponin.

Mouse monoclonal antitubulin antibody (Amer- Of intracellular ca2' antagonists sham) was diluted 1/250 in the Same buffer and

Prophase-arrested oocytes suspended in CFSW applied for 2 h at 37°C. After incubation, slides were incubated in the dark with either 150- were washed in Sorensen-Saponin and incubated 300 p M (2-~(2-bis[Carboxymethyl]-aminod-methyl- 45 min at 37 "C with fluorescein-labelled anti- phenoxy) - methyl] -6- methoxy -8- bis[carboxymeth- mouse antibody (Byosis, dilution 1/250) in Soren- yll-aminoquinolinetetrakis-[acetoxymethyl]ester) Sen-Saponin-BSA. Slides were washed 4-5 times (Quin 2/AM), an intracellular penetrating and in Sorensen buffer containing a 1/2000 dilution of trappable Ca2+ chelator CTsien et al., '881, or with a 0.2% stock solution of blue Evans and mounted 10 pM 8(N,N-diethylamino)-octyl 3,4,5 trimethoxy- in Sorensen-glycerol (1:l)-blue Evans. Controls in- benzoate (TMB 81, a Ca2+ antagonist known to cubated with the second antibody alone gave no stabilize intracellular bound Ca2+ mittenhouse- fluorescence. Simmons and Deykin, '78). After 10 min, 3-ml ali-

Chemicals quot samples of treated or control oocytes, both of which received the same amount of the ethanol or PEG (Mr6,000) was obtained from Serva (Heidel- dimethyl sulfoxide (DMSO) vehicle, were simulta- berg, W. Germany); 1-MeAde, BSA and TMB 8

26 P. GUEKKIER ET AL.

Fig. 1. Effect of intracellular Ca2+ antagonists on shaking- 70 min after shaking. (E) is from the control population and induced maturation of incompetent Asterias rubens oocytes. shows a well-formed first maturation spindle; (F) is from the (A,R) Superficial polar (A) and optical lateral view (B) of GV- population treated with 150 pM Quin 2/AM and exhibits a blocked oocytes showing the exclusion of acidic vesicles from persisting nucleolus. (G,H) Sister batches of oocytes observed the animal area where GV is attached. (C,D) Appearance of 3.5 h after shaking. (G) are from the control population and the female asters within the dispersing nucleoplasm after the exhibit a female pronucleus; (H) were treated with 10 pM TMB oocytes have been shaken for 30 sec (Pictures were taken 5 8 and did not change after GVBD. Scale bar is 20 pm. and 8 min later). (E,F) Aspect of two sister oocytes observed

EVOCATION OF MPF 27

from Sigma; and Quin 2/AM and pronase from Calbiochem (San Diego). Stocks solutions of TMB 8 and Quin 2/AM were 1 mM in ethanol and 25 mM in DMSO, respectively.

RESULTS Shaking us. nucleoplasm microinjection

In an extensive series of experiments, covering three successive breeding seasons, we found re- peatedly that Asterias rubens oocytes, collected at any time, never responded either to nucleoplasm microinjection or increased external Ca2+ concen- tration, whereas some batches of Marthaserias gla- cia& oocytes responded to both stimuli. No strict correlation existed, however, between competence (i.e., the capacity to respond to nucleoplasm injec- tion and Ca2+ sensitivity) since some batches of incompetent oocytes were also found able to re- sume meiosis after Ca2+ stimulation. In contrast, all experimental batches of oocytes, taken from both species, were found to respond readily to the shaking procedure that lead them to reach the female pronucleus stage. This was also the case for Astropecten auranciacus, but not for Orthasterias koehleri, Pisaster ochraceus, or Asterina pectinifera oocytes, which proved unable to complete matura- tion following GV disruption and presented a per- manent nucleolus under these conditions.

Cytology and kinetics of spindle formation When Marthasterias glacialis oocytes are treated

with the hormone or various chemical mimetics inchding increased extracellular Ca2+, GVBD usually occurs within 18 min and no asters or

spindles are formed before that stage. Instead, when both competent and incompetent oocytes of Marthasterias glacialis or Asterias rubens are shaken for only 15-30 sec in SW or CFSW, GVBD occurs without any delay and the female asters appear in the dispersing nucleoplasm within 5-10 min while the nucleolus disappears. This process takes place exactly below the attachment zone (where the GV attaches to the animal pole surface) in a region that we [and others (Otto and Schroe- der, '85)] have shown to contain meiotic female centers and to be devoid of those acidic granules which are particularly numerous in the remaining cortex (Fig. 1A-D). In contrast, when nucleoplasm and cytoplasm of incompetent oocytes from both species were mixed through pressure or microinjec- tion, no female asters or spindle formed and the oocytes retained a n intact nucleolus. It thus ap- pears that the mechanical stress induced by shak- ing produces significant changes in the animal pole cortical domain that are not usually triggered fol- lowing a simple mixing of the nucleoplasm and cytoplasm.

Evidence for increased protein phosphorylation and MPF production in shaken or Cd'

stimulated incompetent oocytes The PEG-induced cell fusion experiments pre-

sented in Table 1 and illustrated in Figure 2 dem- onstrate that the shaking procedure, applied to well-characterized batches of incompetent oocytes of Marthasterias glacialis and Asterias rubens, is as efficient as the hormone or increased external Ca2+ in triggering the appearance of MPF. This factor, which acts within 17-22 min following the

TABLE I . Evidence for MPF production by incompetent starfish oocytesa undergoing maturation under the influencg of shaking or increased

extracellular Ca2+

Stage of the Observed

Species for the maturing partner partner breakdown

Marthasterias Controls (1-MeAde, 1pM) Oocyte 1 616

Modality of GVBD induction arrested nuclear

glacialis Ootid 15115

Increased extracellular Oocyte 1 13/13 calcium (64 mM)

Asterias rubens

Shaking 30 sec Oocyte 1 818

Controls (1-MeAde, 1pM) Oocyte 1 717 Ootid 414

Ootid 818

Shaking 30 sec Oocyte 1 515 Ootid 616

aIncompetent starfish oocytes are oocytes that do not form asters or spindle following microinjection-induced or pressure-induced mixing of their nucleoplasm and cytoplasm. bPEG-induced cell fusions.

28 P. GUERRIER ET AL.

Fig. 2. Appearance of MPF following shaking-induced ootid. (C,D,E) Three steps showing disruption of the female pronucleus envelope as observed 13, 20 and 23 min after fu- sion. Arrows point to the clear area issued from the disrupted GV of the shaken oocyte partner. Scale bar is 20 pm.

meiosis reinitiation in incompetent oocytes of Asterias rubens. (A,€%) Two different focus planes showing polyethylene glycol- induced fusion between a shaken GV-blocked oocyte and an

fusion, was found able to promote nuclear disrup- unaffected following nucleoplasm microinjection. tion both in the GV-arrested oocytes and in the Two possible effectors were considered which might ootids that contained the resting female pronu- have played a role at this level: the arachidonic cleus (Fig. 2). Control experiments showed that acid and the calcium ion. homologous pairs consisting of two oocytes, one (GV stage), or two ootids (female pronucleus stage) never underwent maturation (0/66 and 0/51, re-

Effect of BSA and Cd' antagonists on shaking- induced maturation

spectively). Instead, the heterologous association oocyte l-ootid resulted in the reappearance of MPF activity. Under these conditions, however, nuclear envelope breakdown did not occur before 35-45 min (Guerrier and Nhant, '86). In another experi- ment, we verified that shaking also stimulated protein phosphorylation to the same extent as it does following hormone or extracellular Ca2+ stimulation (Table 2). From these data, one must conclude that shaking does actually activate those MPF precursor molecules which usually remain

Two observations seem to exclude the possibility that shaking may act through the release of ara- chidonic acid in the external medium. First, BSA (10-20 mM), which blocks arachidonic acid-in- duced but not hormone-induced maturation (Mei- jer et al., '86), was found unable to inhibit shaking- induced maturation (Table 3). Second, Astropecten oocytes, which are insensitive to arachidonic acid, usually responded to shaking. In Table 3, external calcium concentration was increased from 10 to about 200 mM by mixing different amounts of

EVOCATION OF MPF

TABLE 2. Changes of the extent of 32P incorporation into proteins as observed on aliquots from the same batch o f incompetent oocytes from

Marthasterias glacialis oocytes submitted to various stimuli"

Conditions of stimulation measure (min) (cpm/mg protein) Maturation

Unstimulated controls 0 16102 + 769 - (GV-arrested oocytes) 20 17790 k 454 -

40 18249 441 -

Hormone-stimulated 40 32036 f 1900 + b Ca2+-stimulated 40 28012 _+ 347 + Shakcn for 30 sec 20 27494 + 1267 +

Time of the "P incorporated

29

"Determinations were made on triplicate samples. b + , Maturation occurred up to female pronucleus formation.

TABLE 3. Response of incompetent starfish oocyte populations to nonhormonal stimuli and test of the effects of BSA (20 mM), Quin 2 (300 pM), and TMB 8 ('10 pM) on shaking-induced maturations

Material Increased Shaking Shaking Shaking

external Ca2+ Shaking +BSA +0uin 2 +TMB 8

Marthasterias glacialis (1)- -= (early season) (2)+ +b

(3)+ + (4)- - (5)+ + (6)+ + (7)- -

Asterias rubens (8)- - (full season) (9)- -

(10)- - (11)- - (12) - - (12)- -

+ + ++ + + ++ ++ ++ i f

++ ++ ++ ++ + +- ++

OC 0 0 ++ + + + + 0 0 0 0 0 + + + +

0 0 0 0 + -d +- + - 0 0 f - +- +- f -

0 0 0 0 0 0 0 0 0 +- +- +- + -

a - - , No induction of GVBD. b+ +, GVBD followed by polar bodies extrusion and formation of female pronucleus. '0, Not tested. d + -, GVBD induction without further evolution.

CFSW and an isoosmotic 0.32 M CaC12 solution. loaded with 150-300 pM Quin-2 AM or 10 pM TMB Batches 3 and 5 reached respectively 70% and 8 (Table 3). They remained blocked after GVBD 100% meiosis at 72 mM Ca2+, batches 2 and 6 50% induction and presented a permanent nucleolus meiosis at 134 and 196 mM respectively, while (Fig. 1E-H). The same drugs, applied for only 30 batches 1, 4 and 7 to 13 were irresponsive at the min and then washed out, were also found to in- same concentration, Mechanical disruption of the hibit the inducing action of 1-MeAde threshold GV was performed by shaking, for 15 to 30 sec, 3 concentrations (Fig. 3). Both effects proved to re- ml aliquots of a n oocyte suspension, which re- versible by adding higher concentrations of the ceived the tested drugs 10 to 20 min before. Sam- hormone to the oocytes (1-100 pM). ples were transferred to a 20 ml bowl and swirled for 20 min in the presence of 0.05% pronase which digested disrupted extracellular investments and In the present work, we confirm that a mechani- avoided clumping of the oocytes. Controls were run cal stress, applied by shaking, could trigger meiosis simultaneously with equivalent concentrations of reinitiation of GV stage-arrested oocytes from at the DMSO or ethanol vehicles. All blocked oocytes least three species of starfish, i.e., Marthasterias resumed maturation when further treated with glacialis, Asterius rubens, and Astropecten auran- excess 1-MeAde (1 to 100 pM). ciacus. Full maturation was also obtained when

In contrast, we found that the same shaken 00- shaking was applied to incompetent oocytes that cytes of Marthasterias glacialis and Asterias r u b could not complete meiosis following pressure-in- ens could not achieve meiosis, when previously duced or microinjection-induced GV disruption.

DISCUSSION

30 P. GUERR :IER ET AL.

acidic vesicles, attaching the GV to the animal pole. It is likely that this activation step involves a localized Ca2+/H+ exchange, as already sug- gested on the basis of experiments pointing to an inhibitory effect of applied weak bases (Doree and Kishimoto, '81; Doree et al. '81, 82). Thus, we sus- pect that nonhormonal stimuli may act quite early at this level, inducing a localized intracellular Ca2+ shift which would be necessary for the acti- vation of both MPF precursor molecules, protein kinases, and female centrioles. These centers, once activated, will be allowed to produce only a spindle at a later stage, provided they are brought in con- tact with the GV nucleoplasm (Schuetz and Longo, '81). This double control mechanism (ionic and nu- clear) would account for both the precocious ap- pearance of the female asters, as observed after shaking-induced GV disruption, and their absence following a simple nucleoplasm microinjection.

It is worth mentioning that a similar dual con- trol seems to exist in the bivalve mollusk Barnea where we observed that a KC1-dependent Ca2+ influx triggered the appearance of the female as- ters slightly before the onset of GVBD. It is likely that the hormone I-MeAde, which has been shown to trigger an early and transient Ca2 -t release from the inner part of the plasma membrane (Guerrier et al., '78; Moreau et al., '78) and to promote corti- cal granule breakdown when applied to later (Pi- card and Doree, '83), may act in the same way to stimulate resting centrioles. This assumption seems to be supported by the fact that prefertilized GV-anucleated oocyte fragments cannot produce male asters before hormone stimulation (Guemier et al., '83; Hirai et al., '81) and will only form disasters (i.e. potential spindles) following a subse- quent nucleoplasm microinjection (Yamada and Hirai, '82). During the course of this work, we also found that the inhibition of shaking-induced and hormone-induced maturations by the Ca2+ antag- onists Quin 2-AM and TMB 8 could be released by increasing 1-MeAde concentration, a process which has been already shown to produce an increased intracellular Ca2+ surge as demonstrated both in vivo (Moreau et al., '78) and in vitro, using an acellular assay system (Doree et al., '78; Moreau and Guerrier, '80).

Even when no similar Ca2+ release could be ob- served following 1-MeAde stimulation of another starfish species (Eisen and Reynolds, '84), a num- ber of experimental evidence still supports our con- clusions. Thus, it has been shown that (1) moderate Ca2+ iontophoresis, but not the ionophore A 23187, may trigger meiosis reinitiation in Marthasterias

P

> (3

m

x $ C o n t r o l s

0 Q u i n 2

T M B B

~~

5 0.5 0.75 1

p M I I - M e A d e l

Fig, 3. Changes in the responsiveness to the hormone of two different batches of Asterius ruhens oocytes which were incubated either with 10 pM TMB 8 (A A) or 300 pM Quin 21 AM (M 0) for 30 min before 1-MeAde addition. Open symbols refer to batch 1; closed symbols to batch 2. Controls (0 0) were run using the same concentration of the ethanol or DMSO vehicle. All these experiments were performed in the absence of external Ca2+, which does not affect the response of the oocytes to the hormone.

This mechanical treatment, which also results in immediate GV disruption, was found to trigger a dramatic increase in the phosphorylation of endog- enous proteins and to activate those MPF precur- sor molecules that usually remain unaffected following a simple nucleoplasm dispersion. Thus, it appears that the GV-blocked oocyte cannot be considered a two compartment system that would contain all the elements required to engage meiosis when these factors will be brought in direct con- tact. This is clearly shown by the fact that shaking remains ineffective when applied to other starfish species and by the failure of injected GV nucleo- plasm to trigger meiosis reinitiation in incompe- tent oocytes.

These findings indicate that female microtubule organizing centers (MTOC), which stand in the cytoplasm outside the GV, must be activated via an early process that is independent of nucleo- plasm diffusion before they can form a spindle. Our experiments involving the use of intracellular Ca2+ antagonists such as Quin 2-AM and TMB 8 suggest that this process may be the result of some subtle changes in the intracellular Ca2+ concen- tration or compartmentalization. This change may take place in the cortical domain, which includes the female centrioles and excludes most of the

EVOCATION OF MPF 31

glacialis (Moreau et al., '78; Moreau and Guerrier, '79); (2) drugs which inhibit or stimulate the hor- mone-induced Ca2 + surge in vivo simultaneously affect the biological response; (3) only active struc- tural analogues and mimetics of the hormone are able to release Ca2+ from a plasma membrane rich fraction obtained from isolated cortices; (4) these in vitro responses could be modulated by the same inhibitor concentrations which proved to be effec- tive in vivo (Dor6e et al., '78; Moreau and Guerrier, '80); and (5) agents known to cause an increase in intracellular Ca2+ will facilitate hormone-induced maturation (Nemoto, '82).

It, thus, appears that a premature Ca2+ shift, arising independently of 1-MeAde stimulation, might account for the appearance of the so-called "competent oocytes" that occur during the late season in Marthasterim glaciulis, while its failure to be triggered would explain those cases of insuf- ficient stimulation that were observed in other spe- cies and resulted in GV-broken oocytes bearing a permanent nucleolus. We would stress that there is no contradiction between this assumption and the well-known observations that tubulin polymer- ization and MPF activity are highly sensitive to increased calcium levels, as observed in vitro, since we already described that ionophore-activated or fertilized GV-blocked starfish oocytes normally reinitiated meiosis after 1-MeAde stimulation (Guerrier et al., '78, '83). More recently, it has even been shown that intracellular Ca2+ surges evoked by fertilization or parthenogenetic activation of starfish oocytes, taken between 30-50 min after hormone stimulation, did not affect the extent of MPF activity or protein phosphorylation found at this stage (Capony et al., '86).

We have ignored for the time being the possibil- ity that the Ca2+ transient we postulate to be necessary might act through the activation of Ca2+-dependent proteases. It remains, however, that various protease inhibitors have been shown to block the activation of MPF precursor molecules both in amphibian and starfish oocyte (Guerrier et al., '77b; Kishimoto et al., '82; Sano and Kanatani, '83). This may open a new area of productive research.

ACKNOWLEDGMENTS We are greatly indebted to Mrs. C. Guerrier for

excellent assistance at every step and to Mrs. N. Guyard for typing the manuscript. This work was partly supported by ATP 84C0939 from the Minis- try of Research and Industry and by the ARC (fel- lowship to I.N.)

LITERATURE CITED Bulet, P., T. Kishimoto, and H. Shirai (1985) Oocyte compe-

tence to maturation-inducing hormone. I. Breakdown of ger- minal vesicles of small oocytes in starfish, Asterina pectinifera. Dev. Growth Differ., 27:243-250.

Capony, J.P., A. Picard, G. Peaucellier, J.C. Labbe, and M. Doree (1986) Changes in the activity of the maturation- promoting factor during maturation and following activa- tion of amphibian and starfish oocytes: their correlations with protein phosphorylation. Devl. Biol., 117rl-12.

Doree, M., and P. Guerrier (1975) Site of action of l-methylad- enine in inducing oocyte maturation in starfish: kinetical evidences for receptors localized on the cell surface. Exp. Cell Res., 96:296-300.

Doree, M., and T. Kishimoto (1981) Calcium mediated trans- duction of the hormonal message in l-methyladenine-in- duced meiosis reinitiation of starfish oocytes. In: Metabolism and Molecular Activities of Cytokinins. J. Guern and C. Peaud-Lenoel (eds). Springer-Verlag, Berlin, pp. 338-348.

Doree, M., P. Guerrier, and N.J. Leonard (1976) Hormonal control of meiosis: specificity of the 1-methyladenine recep- tors in starfish oocytes. Proc. natn. Acad. Sci. U.S.A.,

Doree, M., M. Moreau, and P. Guerrier (1978) Hormonal con- trol of meiosis. In vitro induced release of calcium ions from the plasma membrane in starfish oocytes. Exp. Cell Res.,

Doree, M., K. Sano, and H. Kanatani (1982) Ammonia and other weak bases applied at any time of the hormone depen- dent period inhibit 1-methyladenine-induced meiosis reini- tiation of starfish oocytes. Devl. Biol., 90:13-17.

Doree, M., T. Kishimoto, C. Le Peuch, J.G. Demaille, and H. Kanatani (1981) Calcium-mediated transduction of the hor- monal message in meiosis reinitiation of starfish oocytes. Exp. Cell Res., 135:237-249.

Eisen, A., and G. Reynolds (1984) Calcium transients during early development of single starfish (Asterias forbesi) oo- cytes. J. Cell Biol., 99:1878-1882.

Gerhart, J., M. Wu, and M. Kirschner (1984) Cell cycle dynam- ics of an M-phase-specific cytoplasmic factor in Xenopus lae uis oocytes and eggs. J. Cell Biol., 98:1247-1255.

Guerrier, P., and M. Doree (1975) Hormonal control of reinitia- tion of meiosis in starfish. The requirement of l-methylad- enine during nuclear maturation. Devl. Biol., 47:341-348.

Guerrier, P., and I. NBant (1986) Metabolic cooperation follow- ing fusion of starfish ootid and primary oocyte restores meiotic-phase-promoting activity. Proc. natn. Acad. Sci. U.S.A., 83:48144818.

Guerrier, P., M. Moreau, and M. Dor6e (1977a) Hormonal control of meiosis in starfish: stimulation of protein phos- phorylation induced by 1-methyladenine. Mol. Cell Endocri- nol., 7:137-150.

Guerrier, P., M. Moreau, and M. Doree (1977b) Inhibition de la reinitiation de la meiose des ovocytes de Xenopus Zaeuis par trois antiproteases naturelles, l'antipaine, la chymosta- tine et la leupeptine. C.R. Acad. Sci. Paris, 284:317-319.

Guerrier, P., M. Moreau, and M. Doree (1978) Control of meiosis reinitiation in starfish: calcium ion as the primary effective trigger. Ann. Biol. anim. Bioch. Biophys., 18r441-452.

Guerrier, P., M. Moreau, and M. Dor6e (1979) Relationships between hormone-induced calcium release and "rubidium uptake stimulation in starfish oocytes. Rev. Can. Biol., 38:145-156.

73: 1669-167 3.

115r251-260.

32 P. GUERRIER ET AL.

Guerrier, P., L. Meijer, M. Moreau, and F.J. Longo (1983) Amsterdam, pp. 219-226. Hormone-independent GVBD induces cytoplasmic maturity Moreau, M., and P. Guerrier (1980) In vitro interaction be- in the starfish oocyte. J. Exp. Zool., 226:303-309. tween membrane, hormone and cyclic nucleotides as re-

Hirai, S., Y. Nagahama, T. Kishimoto, and H. Kanatani (1981) vealed with aequorin. Devl. Biol., 79:488492. Cytoplasmic maturity revealed by structural changes in in- Moreau, M., P. Guerrier, and M. Doree (1978) Hormonal con- corporated spermatozoon during the course of starfish oocyte trol of meiosis reinitiation in starfish oocytes. New evidence maturation. Dev. Growth Differ., 23~465-478. for the absence of efficient intracellular receptors for l-meth-

Hiramoto, Y. (1974) A method of microinjection. Exp. Cell yladenine recognition. Exp. Cell Res., 115:245-249. Res., 87.-403-406. Moreau, M., P. Guerrier, M. Doree, and C.C. Ashley (1978) 1-

Kanatani, H. (1973) Maturation-inducing substances in star- methyladenine-induced release of intracellular calcium trig- fishes. Int. Rev. Cytol., 35:253-298. gers meiosis in starfish oocytes. Nature, Lond., 272:251-253.

Kanatani, H., and H. Hiramoto (1970) Site of action of 1- Nemoto, S.I. (1982) Nature of the 1-methyladenine-requiring methyladenine in inducing oocyte maturation in starfish. phase in maturation of starfish oocytes. Dev. Growth Differ., Exp. Cell Res., 61:280-284. 24:429-442.

Kanatani, H., and H. Shirai (1967) In vitro production of Otto, J.J., and T.E. Schroeder (1984) Microtubules arrays in meiosis inducing substance by nerve extract in ovary of the cortex and near the germinal vesicle of immature star- starfish. Nature, Lond., 216:284-286. fish oocytes. Devl. Biol., 101:274-281.

Kanatani, H., H. Shirai, K. Nakanishi, and T. Kurokawa Paweletz, N., D. Mazia, and E.M. Finze (1984) The centrosome (1969) Isolation and identification of meiosis inducing sub- cycle in the mitotic cycle of sea urchin eggs. Exp. Cell Res.,

Kishimoto, T. (1986) Microinjection and cytoplasmic transfer Picard, A,, and M. Dor6e (1983) Hormone-induced partheno- in starfish oocytes. In: Methods in Cell Biology, 27. T.H. genetic activation of mature starfish oocytes. Exp. Cell Res.,

Kishimoto, T., and H. Kanatani (1976) Cytoplasmic factor re- Picard, A., and M. Doree (1984) The role of the germinaI sponsible for germinal vesicle breakdown and meiotic mat- vesicle in producing maturation-promoting factor (MPF) as uration in starfish oocyte. Nature, Lond., 260:321-322. revealed by the removal and transplantation of nuclear ma-

Kishimoto, T., M.L. Cayer, and H. Kanatani (1976) Starfish terial in starfish oocytes. Devl. Biol., 1043357-365. oocyte maturation and reduction of disulfide-bond on oocyte Rittenhouse-Simmons, S., and D. Deykin (1978) The activation surface. Exp. Cell Res., 101:104-110. by Ca2+ of platelet phosphilipase A,. Effects of dibutyryl

Kishimoto, T., S. Hirai, and H. Kanatani (1981) Role of ger- cyclic adenosine monophosphate and 8-m, N-dimethyl- minal vesicle material in producing maturation-promoting amino~octyl-3,4,5-trimethoxybenzoate. Bioch. Biophys. Acta, factor in starfish oocyte. Devl. Biol., 81~177-181. 543:409422.

Kishimoto, T., T.G. Clark, H.K. Kondo, H. Shirai, and H. Sano, K., and H. Kanatani (1983) Effect of various protease Kanatani (1982) Inhibition of starfish oocyte maturation by inhibitors on starfish oocyte maturation. Biomed. Res., some inhibitors of proteolytic enzymes. Gamete Res., 5:11- 4:139-146. 18. Schroeder, T.E. (1985) Cortical expressions of polarity in the

Lowry, O.M., N.J. Rosenbrough, A.L. Farr, and R.G. Randall starfish oocyte. Dev. Growth Differ., 27:311-321. (1951) Protein measurement with the Folin phenol reagent. Schuetz, A.W., and J.D. Biggers (1967) Regulation of germinal J. Biol Chem., 193:265-275. vesicle breakdown in starfish oocytes. Exp. Cell Res., 46.-624-

Masui, Y., and C.L. Markert (1971) Cytoplasmic control of 628. nuclear behaviour during meiotic maturation of frog 00- Schuetz, A. W., and F.J. Longo (1981) Hormone-cytoplasmic cytes. J. Exp. Zool., 177:129-146. interactions controlling sperm nuclear decondensation and

Meijer, L., and P. Guerrier (1983) Immobilized methylglyoxal- male pronuclear development in starfish oocytes. J. Exp. bis(guany1hydrazone) induces starfish oocyte maturation. Zool., 215:107-111. Devl. Biol., 100:308-317. Shapiro, H. (1941) Centrifugal elongation of cells, and some

Meijer, J., and P. Guerrier (1984) Maturation and fertilization conditions governing the return to sphericity and cleavage in starfish oocytes. Int. Rev. Cytol., 86:129-196. time. J. Cell Comp. Physiol., 18:61-78.

Meijer, L., P. Guerrier, and J. Maclouf (1984) Arachidonic acid, Tsien, R.Y., T. Pozzan, and T.J. Rink (1982) Calcium homeo- 12- and 15-hydroxy eicosatetraenoic acids, eicosapentaenoic statis in intact lymphocytes: cytoplasmic free calcium moni- acid, and phospholipase A2 induce starfish oocyte matura- tored with a new intracellularly trapped fluorescent tion. Devl. Biol., 106.-368-378. indicator. J. Cell Biol., 94:325-334.

Meijer, L., J. Maclouf, and R.W. Bryant (1986) Arachidonic Vassetsky, S.G., G.G. Sekirina, M.N. Skoblina, and A.A. Bil- acid metabolism in starfish oocytes. Devl. Biol., 114:22-33. inkis (1983) The fusion of oocytes of the starfish Aphelaster-

Meijer, L., P. Pondaven, P. Guerrier, and M. Moreau (1984) A iasjaponica. The capacity of cell hybrids for maturation and starfish oocyte user's guide. Cah. Biol. Mar., 25457-480. cleavage. Cell. Differ., 123'3-76.

Moreau, M., and P. Guerrier (1979) Free calcium changes Yamada, H., and S. Hirai (1982) Role of contents of the ger- associated with hormone action in oocytes. In: Detection and minal vesicle in male pronuclear development and cleavage Measurement of Free Ca2+ in Cells. C.C. Ashley and A.K. of starfish oocytes. Dev. Growth Differ., 26:479-487. Campbell (eds). Elsevier North Holland Biomedical Press:

stance in starfish. Nature, Lond., 211~273-274. 152:47-65.

Schroeder (ed). Academic Press, New York, pp. 379-394. 145:315-323.